Abstract Background: Knowledge about Alburnoides remains lacking relative to many other species, resulting in a lack of a systematic position and taxonomic diagnosis. Basic biological information for Alburnoides has been constructed, and it is necessary to understand further and obtain more information about this species. Its phylogenetic relationships are still debated and no molecular data have been used to study this taxon in Iran. A holistic approach for genetic methods was adopted to analyze possible spirlin population differences at selected centers in the south Caspian Sea basin of Iran. Methods: The phylogenetic relationships were determined based on 774 base pairs of the mitochondrial cytochrome b gene of 32 specimens of spirlin from nine locations in the south Caspian Sea drainage basin of Iran. The nucleotide sequences were subjected to phylogenetic analysis using the neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian methods. Results: The mitochondrial gene tree largely supports the existence of three major clades. The western populations (clade I) may be considered as Alburnoides eichwaldii, whereas the Talar river populations (clade II) are represented as Alburnoides sp.1 and the eastern populations (clade III) may be distinct taxa of Alburnoides sp.2. Conclusion: This molecular evidence supports the hypothesis that A. bipunctatus does not exist in the south Caspian Sea basin of Iran, and that the western and eastern populations are distinct taxa.

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