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Two species of cambarids have been reported for the Yucatan Peninsula, Procambarus llamasi and P. pilosimanus, the latter with a distribution that stretches from the south of Veracruz to the south of Campeche. However, during the last decade only the presence of P. llamasi has been reported; as a consequence, and also because of the biogeographic isolation of the Laguna de Chichancanab, we here investigated two southern populations, Procambarus sp. from Laguna de Chichancanab, Quintana Roo (LCh) and Procambarus llamasi from Ignacio Zaragoza, Campeche (IZa). We investigated these at the molecular level in order to determine if they are independent genetic units. The levels of genetic heterogeneity of the mitochondrial r16S and subunit I of the cytochrome oxidase (CO-I) genes are thus evaluated and contrasted to the levels of the cambarellinid, Cambarellus montezumae from the Xochimilco Channels, D.F. (XCh). The phylogenetic relations of these populations in relation to other Neartic and Neotropical astacoids (Astacidae and Cambaridae) and Caribbean atyids (Atyidae) were investigated. Levels of genetic diversity (h and π) in both r16S and CO-I were lower in the southeastern populations in relation to the Cambarellinae from central Mexico. For instance, π ranged from 0.00000 to 0.00149 for r16S and CO-I, respectively, in the southeastern populations, whereas the central population showed levels that ranged from 0.00250 to 0.00232 for the same gene regions. Both gene regions indicate significant levels of structuring among the southeastern populations (FST, 0.714 and 0.612, P < 0.0001, for r16S and CO-I), and these results are supported by the lack of actual gene flow. However, in order to confirm this hypothesis a larger number of populations along the range of distribution of the species should be tested. The phylogenetic reconstruction by maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) of these populations and other astacids and atyids, confirms the monophyly of the two southeastern populations (P. llamasi) as well as of C. montezumae, and suggests that Cambaridae constitutes a non-monophyletic assemblage, a hypothesis herein confirmed by a total evidence Bayesian analysis. [ABSTRACT FROM AUTHOR]