Abstract: Puntius tetrazona is a small tropical ornamental fish with high economic value. In this study, the process of embryonic development was observed. The chromosome number and DNA content of P. tetrazona were detected with the small fish swimming method and the flow cytometry. Meanwhile, the phylogenetic analysis of the Barbinae subfamily was performed with mitochondrial genome sequences. The results showed that the embryonic development process of P. tetrazona was classified into 7 stages, including the fertilized egg stage, cleavage stage, blastocyst stage, gastrula stage, neurula stage, organogenesis stage and hatching stage. Compared to zebrafish which hatched out 48 hours post fertilization (hpf) with pigment, P. tetrazona hatched 24 hpf at the water temperature of 28 °C without pigment. The chromosome number and DNA content of P. tetrazona are 2n=50 and 1.50 pg/2c, respectively. The phylogenetic tree showed that P. tetrazona appeared earlier during the speciation of the Barbinae subfamily. These results will provide the basis for P. tetrazona breeding and genetic improvement.