Aquaculture activities are steadily expanding in Vietnam, covering an estimated 700,000 ha, with 89% of these culture ponds located in the Mekong Delta. Since 2009, large-scale bacterial epidemics have spread in response to this intensive cultivation. Antibiotics, even those considered as a last resort, have only partially mitigated this problem. In turn, the side effects of the massive use of these chemicals include the appearance of mobile genetic elements associated with antibiotic resistance genes (ARGs). The large-scale emergence of a diverse bacterial resistome, along with severe economic losses, has posed significant health risks to local residents. In this study, the seasonal and spatial distributions of the class I integrase (CL1) intl1 and the ARGs sul2 (sulfonamide), BLA-oxa1 (ß-lactams), and ermB (erythromycin) were quantified from water and sediment samples collected during two consecutive seasons along the Vam Co River and its tributary (Long An province, Vietnam). The results showed that CL1 was present in all river compartments, reaching 2.98×104 copies/mL and 1.07×106 copies/g of sediment, respectively. The highest relative copy abundances to the 16S rDNA gene were measured in water samples, with up to 3.02% for BLA-oxa1, followed by sul2 (1.16%) and ermB (0.46%). Strong seasonal (dry season vs. rainy season) and spatial patterns were recorded for all resistance genes. Higher amounts of ARGs in river water could be associated with higher antibiotic use during the rainy season. In contrast, higher amounts of ARGs were recorded in river sediments during the dry season, making this habitat a potential reservoir of transient genes. Finally, the observations made in this study allowed us to clarify the environmental and anthropogenic influences that may favor the dispersal and persistence of ARGS in this riverine ecosystem.