Zooplankton play a pivotal role in sustaining the majority of marine ecosystems. The distribution patterns and diversity of zooplankton provide key information for understanding the functioning of these ecosystems. Nevertheless, due to the numerous cryptic and sibling species and the lack of diagnostic characteristics for immature developmental stages, the identification of the global-to-local patterns of zooplankton biodiversity and biogeography remains a challenge in different research fields. Here, the spatial and temporal changes in the zooplankton community from the open waters of the southern section of the Gulf of Mexico were assessed using a multilocus sequence analysis and metabarcoding approach based on the genetic information of 18S and cytochrome oxidase c subunit I (COI) genes. Additionally, a multi-scale analysis was implemented to evaluate which environmental predictors may explain the variability in the structure of the zooplankton community. Our finding suggests that the synergistic effects of oxygen, temperature, and longitude (intended as a proxy for still unexplored forces) may explain both spatial and temporal changes in the zooplankton community. Furthermore, the zooplankton distribution likely reflects the coexistence of three heterogeneous ecoregions and a bio-physical partitioning of the studied area. Finally, some taxa were either exclusive or predominant with either 18S or COI data. This may suggest that comprehensive assessments of the zooplankton community may be more accurately met by the use of multi-locus approaches.