The morphology and genetic identification of Rasbora lateristriata and Rasbora argyrotaenia between cultivated and wild populations has never been reported. This study compares morphology and cytochrome c oxidase (COI) genes between farmed and wild stock Rasbora spp. in Java and Sumatra island, Indonesia. We analyzed the truss network measurement (TNM) characters of 80 fish using discriminant function analysis statistical tests. DNA was extracted from muscle tissue of 24 fish specimens, which was then followed by polymerase chain reaction, sequencing, phylogenetic analysis, fixation index analysis, and statistical analysis of haplotype networks. Basic Local Alignment Search Tool analysis validated the following species: R. lateristriata and R. argyrotaenia from farming (Jogjakarta); Rasbora agryotaenia (Purworejo), R. lateristriata (Purworejo and Malang), Rasbora dusonensis (Palembang), and Rasbora einthovenii (Riau) from natural resources. Based on TNM characters, Rasbora spp. were divided into four groups, referring to four distinct characters in the middle of the body. The phylogenetic tree is divided into five clades. The genetic distance between R. argyrotaenia (Jogjakarta) and R. lateristriata (Malang) populations (0.66) was significantly different (p < 0.05). R. lateristriata (Purworejo) has the highest nucleotide diversity (0.43). R. argyrotaenia from Jogjakarta and Purworejo shared the same haplotype. The pattern of gene flow among them results from the two populations' close geographic proximity and environmental effects. R. argyrotaenia had low genetic diversity, therefore, increasing heterozygosity in cultivated populations is necessary to avoid inbreeding. Otherwise, R. lateristriata (Purworejo) had a greater gene variety that could be used to develop breeding. In conclusion, the middle body parts are a distinguishing morphometric character of Rasbora spp., and the COI gene is more heterozygous in the wild population than in farmed fish, therefore, enrichment of genetic variation is required for sustainable Rasbora fish farming.