Abstract The main drivers of diversification of freshwater fishes in Cuba are not yet well understood. For example, salt tolerance was thought as the main factor involved in the diversification of Gambusia punctata species group in this archipelago. However, evidence from a recent DNA barcoding survey suggested the presence of cryptic species and no correlation between species delimitation and level of salinity. In this study, we analyzed the cryptic diversification of G. punctata species group in Cuba, based on a comprehensive sampling of its distribution and including habitats with different salinity levels. We evaluated the patterns of molecular divergence of the samples by sequencing a set of mitochondrial DNA (mtDNA) regions and genotyping nine nuclear microsatellite loci. We also used cytochrome b gene ( cyt b) partial sequences and these microsatellite loci to analyze population structure inside putative species. Five mtDNA well-differentiated haplogroups were found, four of them also identified by the analysis of the microsatellite polymorphism which corresponds to two already recognized species, G. punctata , and G. rhizophorae , and three putative new species. The extent of hybrid zones between these groups is also described. In each group, populations inhabiting environments with contrasting salinity levels were identified, indicating a generalized trait not specific to G. rhizophorae . The geographic distribution of the groups suggested a strong association with major relict territories of the Cuban Archipelago that was periodically joined or split-up by changes in seawater levels and land uplifts. Salinity tolerance might have facilitated sporadic and long-distance oversea dispersal but did not prevent speciation in the Cuban archipelago.