The study dealt with the standardization of sperm cryopreservation protocol of Indian Major Carp, Rohu (Labeo rohita) to establish a cryogenic sperm bank and production of quality seeds using cryopreserved sperm in commercial fish hatcheries. A series of experiments were conducted to achieve the goal. Quality broodstock of Rohu was developed by collecting and rearing of Halda and Padma river-origin fish in earthen ponds with supplementary feeds. Activation of sperm motility was evaluated in different gradients of NaCl solution (0.1% to 1%), and highest sperm motility of 96.0±1.0% and swimming duration of 37.46±0.8 min was observed in 0.4% NaCl solution. The toxicity of cryoprotectants (DMSO and methanol) to sperm was tested at different concentrations (5%, 10% and 15%) and incubation time (5–40 min) with two extenders (Alsever’s solution and egg-yolk citrate), and found that cryoprotectants with 5% and 10% concentrations produced better motility during 5 and 10 min of incubation, but 15% concentration seemed toxic to sperm. Alsever’s solution plus 10% DMSO at 1:9 dilution ratio (sperm : diluent) with 10 min equilibration time and 10°C/min cooling rate showed best equilibration motility (94.3±2.3%) and post-thaw motility (90.0±2.9%) in cryogenic freezing. Post-thaw motility of cryostored sperm remained at satisfactory level (initial 90±0% and final 81.67±0.33%) during assessment over the twelve-month storage period. During induced breeding in 11 public and private hatcheries, the average fertilization and hatching rates were estimated as 40.41±3.33% and 32.53±3.07% with cryopreserved sperm, whereas 75.22±3.85% and 63.23±4.56% with hatchery-origin fresh sperm (control) respectively. The seeds of both cryopreserved sperm-origin and controls were reared in the respective hatcheries and sampled monthly to compare their growth performances. The average final length and weight of cryopreserved sperm-origin and control fry were found as 16.76±4.11cm, 77.79±52.49g and 11.56±0.24cm, 15.28±1.19g respectively after 180 days pond rearing. Cryopreserved sperm-originated fry showed significantly (p<0.05) higher growth than those of fresh sperm due to introduction of new superior germplasm through artificial insemination using cryopreserved sperm collected from the cryogenic sperm bank. The results also indicate that the hatchery stocks become genetically inferior due to inbreeding, negative selection and poor broodstock management, and thus demands for improvement of quality of seeds and broodstocks through sperm cryopreservation technology.