For the initiation of a genetic improvement program of any culturable fish species, the assessment of the genetic quality of different stocks is prerequisite. However, in Pakistan, such data of all culturable fish species including an indigenous Labeo rohita are lacking. Here, an attempt has been made to determine the genetic diversity among five different stocks of L. rohita using DNA barcoding with cytochrome oxidase subunit I (COI) and cytochrome b genes and then compared their growth performance, immune response, and disease resistance. For the evaluation of growth performance, fingerlings of five different stocks; TH-S (Tawakkal Fish Hatchery Muzaffargarh), MCH-S (Government Fish Hatchery Mian Channu), RH-S (Rawal Fish hatchery Islamabad), SH-S (Farooqabad Fish Hatchery Sheikhupura), and MRH-S (Char Banda Fish Hatchery Mardan) across Pakistan were collected, PIT tagged, and reared in communal earthen ponds for 3 months under semi-intensive culture conditions. Similarly, for the evaluation of immunity and disease resistance, the environmental influence was minimized by conducting experiments under control conditions and challenging with Staphylococcus aureus. The results of both DNA barcoding markers indicated the significant level of genetic difference among different stocks, as clades in the phylogenetic trees clearly separated five stocks. The growth performance, immunity, and disease resistance data also revealed significant (p < 0.05) differences among them, as TH-S followed by MCH-S showed the highest growth performance in term of weight gain, specific growth rate, and the expression of Insulin like growth factor II (IGF II) and Preproghrelin genes in muscle and liver tissues compared to other strains. The post-challenge survival (%), and the expression of immune-related genes, i.e., Natural killer enhancing factor (NKEF), Complement component (C9), Hepcidin, and Transferrin genes also showed a similar trend among strains. The overall results indicated the genetic variability and variation in growth performance and disease resistance among different stocks of L. rohita collected from different hatcheries. Based on the results, it seems that TH-S has more vigor in terms of growth rate and disease resistance.