The Mekong River holds global significance as a vital source of diverse aquatic resources, essential for sustaining livelihoods in the Mekong basin. This study focused on the fish species diversity at 16 sampling sites in the northeastern part of the Mekong River in Thailand, with data collected monthly from October 2013 to September 2014. A total of 164 fish species, belonging to 11 orders, 32 families, and 97 genera, were identified. The dominant family, Cyprinidae, included 74 species (45.1%). Notably, 153 species were found across all surveyed areas, while 6.7% (11 species) were exclusive to specific locations. Four species—Parambassis wolffii (Bleeker, 1850), Urogymnus polylepis (Bleeker, 1852), Scaphognathops stejnegeri (Smith, 1931), and Pangasius sanitwongsei (Smith, 1931)—displayed limited distribution, observed in less than 30% of the surveyed area. This limited distribution is likely due to various alterations to the Mekong River, highlighting the urgent need for conservation efforts to address the potential extinction risk facing these species.