An experiment was undertaken to find the most appropriate explant type, (i.e., shoot tip, leaf, immature fruit and anther explants) and the appropriate growth regulator concentrations and combinations on callus formation and shoot regeneration in 3 strawberry (Fragaria x ananassa Duch.) cultivars, namely Festival, Fortuna and Sweet Charlie. As for callus formation in response to growth regulators, ten combinations between 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 6-Benzylaminopurine (BAP) were used. significant variances among different media were obtained and 0.5mg L^-1 BAP +1.5 mg L^-1 2, 4-D produced the highest response (95.75%) in comparison to other media. Maximum callus formation percentage was obtained the using runner explant (75.38%) while immature fruit explant gave the lowest value. Festival and Fortuna cultivars recorded higher percentage of callus formation than Sweet Charlie. Calli derived from the good callus induction medium were tested for shoot regeneration with ten different combinations from BAP and NAA. The results exhibited that using runner as explant source and 2.0 mg L^-1 BAP with the three tested cultivars recorded significant regeneration percentage in comparison to other combinations.