The negative ramifications of invasive alien species (IAS) are considered the second-most cause of biodiversity extinction and endangerment after habitat modification. IAS movements are mainly anthropogenically driven (e.g., transport of shipping containers) and require fast detection to minimize damage and cost. The present study is the first to use molecular biosurveillance of international shipping containers to detect IAS and regulated species identification in Canada. Thirty-eight samples were collected from debris (soil, stems, seeds, individual specimens) found in containers arriving in Canada. A multi-marker approach using COI, ITS, ITS2, and 16S was used to identify four main taxonomic groups: arthropods, fungi, plants, and bacteria, respectively. Eleven IAS species were identified via metabarcoding based on environmental DNA samples, including two arthropods, six fungi, two plants, and one bacteria. The origin of the eDNA detected from each species was linked to their native distribution and country of origin, except for Lymantria dispar. Four physical specimens were also collected from shipping container debris and DNA barcoded, identifying three non-regulated species (two arthropods and one fungus). Altogether, these results demonstrate the importance of integrating molecular identification into current toolkits for the biosurveillance of invasive alien species and provide a set of validated protocols ready to be used in this context. Additionally, it reaffirms international shipping containers as a pathway for multiple invasive aliens and regulated species introduction in Canada. It also highlights the need to establish regular and effective molecular biosurveillance at the Canadian border to avoid new or recurrent invasions. Las ramificaciones negativas de las especies ex & oacute;ticas invasoras (EEI) se consideran la segunda causa de extinci & oacute;n y peligro de la biodiversidad despu & eacute;s de la modificaci & oacute;n del h & aacute;bitat. Los movimientos de EEI son impulsados principalmente por causas antropog & eacute;nicas (por ejemplo, transporte de contenedores de env & iacute;o) y requieren una detecci & oacute;n r & aacute;pida para minimizar da & ntilde;os y costos. El presente estudio es el primero en utilizar biovigilancia molecular de contenedores de env & iacute;o internacionales para detectar EEI y la identificaci & oacute;n de especies reguladas en Canad & aacute;. Se recolectaron treinta y ocho muestras de material (tierra, tallos, semillas, espec & iacute;menes individuales) encontrados en contenedores que llegaron a Canad & aacute;. Se utilizaron m & uacute;ltiples marcadores moleculares, COI, ITS, ITS2 y 16S, para identificar cuatro grupos taxon & oacute;micos principales: artr & oacute;podos, hongos, plantas y bacterias, respectivamente. Se identificaron once especies de EEI mediante matabarcoding basado en ADN ambiental, incluidos dos artr & oacute;podos, seis hongos, dos plantas y una bacteria. El origen del ADN ambiental detectado de cada especie estuvo vinculado a su distribuci & oacute;n nativa y pa & iacute;s de origen, excepto Lymantria dispar. Tambi & eacute;n se recolectaron cuatro espec & iacute;menes en los contenedores de env & iacute;o y se analizaron mediante c & oacute;digo de barras de ADN, identificando tres especies no reguladas (dos artr & oacute;podos y un hongo). En conjunto, estos resultados demuestran la importancia de integrar la identificaci & oacute;n molecular dentro de las herramientas actuales para la biovigilancia de especies ex & oacute;ticas invasoras y proporcionan un conjunto de protocolos validados listos para ser utilizados en este contexto. Adem & aacute;s, reafirma que los contenedores de transporte internacional son una v & iacute;a para la introducci & oacute;n de m & uacute;ltiples especies ex & oacute;ticas invasoras y especies reguladas en Canad & aacute;. Tambi & eacute;n destaca la necesidad de establecer una biovigilancia molecular peri & oacute;dica y eficaz en la frontera canadiense para evitar invasiones nuevas o recurrentes.