Extensive chromosome variation involving Robertsonian and non-Robertsonian changes were proposed to explain chromosomal evolution within killifishes of the aplocheiloid group belonging to the order Cyprinodontiforms. In the present work we describe the karyotypes of four Cynolebias species and analyze chromosome changes by means of mitochondrial phylogenetic studies, including 10 taxa of this genus. Diploid numbers varied from 48 to 44 and the number of chromosome arms from 50 to 54. Molecular phylogenetic analyses allow us to corroborate previous hypothesis about chromosome evolution in aplocheiloid fishes. The tree topology based on a combined dataset of mitochondrial cytochrome b and 12S genes shows that recent cladogenetic events within the genus Cynolebias could have occurred by allopatric or 'in-situ' differentiation involving chromosomal rearrangements. Our analyses of approximately 10% of mitochondrial genome can be helpful in determining these recent cladogenetic events but it showed limited phylogenetic resolution at intermediate levels of divergence. This can be explained in part by the high levels of DNA sequence divergence (ranging from 0.015 to 0.245) detected at intrageneric level. Different methodological approaches suggest that chromosomal changes in Cynolebias have occurred during their differentiation, supporting the hypothesis that the unresolved basal polytomy could be the result of rapid speciation events, like a true 'star polytomy'.