Plant tissue culture is one of the most efficient biotechnological techniques, which reduces time, effort, space, and finance needed for plant improvement. Here, the effect of genotype and different combinations of plant growth regulators (PGR) on the callus formation of sweet basil (Ocimum basilicum L.) was investigated. Twelve callus induction media, supplemented by different combinations of 6-Benzylaminoburine (BAP) and Naphthalene acetic acid (NAA), were tested with two basil landraces (i.e., Balady and French). Results showed highly significant differences among the PGR and basil landraces, as well as their interaction. Balady was superior in all evaluated traits to the French landrace. The percentage of callus formation ranged from 37.47 to 100%. The callus fresh weight per explant (CFW/exp) in Balady ranged from 159.49 to 1275.00 mg obtained by 20 and 10_M BAP, respectively. Meanwhile, the CFW/exp for the French landrace ranged from 115.74 to 446.29 mg obtained by 10_M NAA and 40_M BAP + 5_M NAA, respectively. The callus dry weight per explant (CDW/exp) for Balady ranged from 17.94 to 82.08 mg formed by 20_M BAP and 10_M + 10_M NAA, respectively. On the other hand, the highest CDW/exp for the French landrace was 42.78 mg obtained by 20_M BAP + 10_M NAA, while the lowest CDW/exp was 4.04 mg resulted by 10_M NAA. These results indicate that callus formation in basil is significantly influenced by both genotype and PGR. Thus, using the appropriate type and combination of PGR for each genotype can effectively enhance the callus formation rate in basil.