Turkey has a rich freshwater biodiversity in terms of Cyprinid genus in respect to its geographical location. To elucidate the phylogeny of the Alburnoides genus, one of these genera, genetic data for the cytochrome b gene (1141 bp) was generated for 445 samples collected at 42 sampling sites across their geographical distribution. A total of 54 mitochondrial haplotypes identified were distrubuted among distinct twelve species that did not share haplotypes with each other. Pairwise sequence divergence among these species range from 1.37% (A. emineae and A. velioglui) and 10.99% (A. manyasensis and A. smyrnae). A new potential species in the River Dirgine that run into the Black Sea Basin was separated from the most closed known species with mean 6.3%. Network analysis and phylogenetic analysis indicated that all haplotypes were clustered into two major clades, which corresponded to twenty-three Alburnoides lineages, with moderate-high bootstrap supports and mutational steps, respectively. Application of a molecular clock to a Bayesian phylogeny indicates that Alburnoides diversified under the paleogeographic conditions such as tectonic uplift and faulting Miocene aged as well as climatic oscillation and sea-level fluctuations during late Miocene-middle Pleistocene. The genetic results of the present study indicated the inter-specific distance of cyt b gene sequences followed the ideal results for species identification and phylogeny of Turkish spirlins.