Puntius spp has the largest number of species in the Asian tropics with some unsolved taxonomic problems at the species level. The phylogenetic relationships are inferred from DNA sequences of the three mitochondrial genes (Cytochrome oxi- dase b (875 bp), Cytochrome oxidase c (498 bp), 16s ribosomal RNA (574) and two nuclear genes, Recombination activating gene 2 (863 bp) and Beta actin (913 bp). A multi-locus molecu- lar phylogeny was constructed and calibrated using two fossil dates to estimate divergence times within the Cyprinid family. The topologies resulting from maximum parsimony, maximum likelihood and Bayesian analyses of combined genes are broad- ly congruent with each other. Two distinct clades within the genus were obtained with high bootstrap values, namely Clade I (P. schwanenfeldii , P. bulu , P. gonionotus , and P. daruphani) and Clade II (P. fasciatus , P. tetrazona hexazona , P. tetrazona partipentazona , P. lateristriga , P. binotatus and P. everetti). Divergence times estimated from nuclear and mitochondrial DNA data calibrated in the data set showed a clear history of diversification over the last 47.87 million years ago (MYA) for cyprinid, and 26.85 (MYA ) for Puntius and supported early Oligocene origins for this genus.

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