Two species of mud crab, Scylla olivacea and Scylla paramamosain, occur sympatrically in Ban Don Bay, Surat Thani Province, Thailand, with no evidence of interspecific hybridization. These crabs are part of a traditional and largely unregulated fishery within Ban Don Bay. As part of a study of their reproductive biology and the possible mechanisms explaining their lack of hybridization, size at first maturity was compared between females of the two species. Female S. olivacea and S. paramamosain were collected from Ban Don Bay, and their size at first maturity was estimated using three methods relating abdomen width to body size data: the minimum size at maturity, breakpoint analysis, and probit analysis. All three methods employed revealed that female S. paramamosain attain a larger mean size at maturity than female S. olivacea by as much as 18 to 30 mm internal carapace width (ICW). Although the mean size at maturity is different between species, there is an overlap in the size at maturity between the smallest mature S. paramamosain (ICW = 101 mm) and the largest immature S. olivacea (ICW = 118 mm). Scylla paramamosain has a generally larger body size than S. olivacea; therefore, it is possible that there is a relationship between body size and the size of functional maturity that prevents hybridization between these two species. However, the overlap in the size at maturity observed between the two species means that there is a margin where size could not be the only isolating mechanism, and therefore, other prezygotic isolating mechanisms must also be responsible for maintaining the sympatric species observed. Based on these findings, it is suggested that the local fishery should operate with a minimum legal size of 100 mm and 130 mm ICW for S. olivacea and S. paramamosain, respectively.