The Pacific white shrimp (Penaeus vannamei) holds significant economic value in Mexican and global aquaculture. However, high production rates, suboptimal farming practices, and environmental factors have increased the prevalence of diseases affecting shrimp populations. Conventional molecular diagnostics, such as PCR and qPCR, facilitate pathogen detection by targeting specific nucleic acids. Despite their utility, these methods cannot simultaneously identify multiple pathogens present in a single sample. Next-generation sequencing (NGS) has emerged as a promising tool for detecting entire microbial communities in environmental samples (e.g., water, sediment), offering enhanced pathogen identification capabilities. This study evaluated the sensitivity of NGS in detecting DNA-based pathogens in P. vannamei from two shrimp farms in Northwest Mexico. Conventional PCR identified several pathogens, including infectious hypodermal and hematopoietic necrosis virus (IHHNV), shrimp hemocyte iridescent virus (SHIV), Baculovirus penaei (BP), Candidatus Hepatobacter penaei (NHP-B), and Vibrio parahaemolyticus (AHPND agent) (Figure 1). Sanger sequencing confirmed these findings. In contrast, NGS only detected AHPND, likely due to low DNA concentrations in water and sediment samples and the limitations of whole-genome amplification reagents. This is the first study to apply a universal PCR protocol for multi-pathogen detection in shrimp aquaculture.