Squash (Cucurbita pepo L.) is highly polymorphic in nature which could contribute its different regeneration potentiality for different genotypes. The present investigation was carried to know the effects of plant growth regulators, explant types, explants age and AgNO3 on in vitro regeneration potentiality of hybrid squash genotype and to optimize regeneration protocol. In this protocol, cotyledon and hypocotyl segments were grown on MS medium supplemented with 1.5 mg/l 6-Benzylaminopurine (BA) and 0.2 mg/l α-Naphthalene acetic acid (NAA), successfully induced callus and subsequently shoots for C. pepo L. cv. First Runner. The height percentage of shoot production frequency was 66.7% from 6 days old cotyledon explants in MS + 1.5 mg/l BA + 0.2 mg/l NAA combination. The shoot regeneration frequency was increased up to 86.67% when 2.0 mg/l AgNO3 was added with MS + 1.5 mg/l BA + 0.2 mg/l NAA media combination by using 5 days old cotyledon explants. Shoot regeneration potentiality of four other C. pepo genotypes were also investigated with this system and showed a substantial amount of regeneration potentiality for different genotypes. Regenerated shoots induced the height 93.33% rooting frequency which promoted long, thick roots on 10 to 12 days in MS medium supplemented with 0.1 mg/l NAA. The regenerated plantlets with long and thick roots were acclimatized in pot soil and eventually grown in natural environment with culture facility. The present study describes a simple and efficient protocol for in vitro plant regeneration of hybrid C. pepo genotypes and may be utilized for further transgenic development.