Abundance estimates for the Antillean manatee, Trichechus manatus manatus, in the southern Gulf of Mexico are based on interviews, mortality reports, and opportunistic sightings. The lack of quantitative data is explained by the complexity of hard-to-access fluviallake systems, low water transparency, and the elusive behavior of manatees. Side-scan sonar is a promissory tool to detect and count manatees in fluvial systems. The Pantanos de Centla Biosphere Reserve (PCBR), in the coastal plain of Tabasco (Mexico), could play a key role in the regional conservation of manatees, but it is under constant pressure, especially from the oil and gas industry, and it is important to generate quantitative data for this area. The objective of this study was to quantify the relative abundance of manatees at 4 selected sites within PCBR and at 2 reference sites outside the reserve using side-scan sonar, as a basis for the long-term monitoring of the species and to determine the importance of the reserve for regional manatee conservation. We conducted 5 to 7 boat surveys on 10-km line transects along selected water courses and recorded 63 manatee sightings. Manatees occurred at all sampling sites and abundance increased upriver. The site with the highest mean abundance (5.17 ± 1.9 manatees/10 km) was located at the southeastern end of PCBR. There was statistical difference among sites. The southeastern portion of Tabasco is a key region for manatee conservation and management in Mexico. PCBR is a key regional feature but it is necessary to define protection strategies within and beyond its southern limits.