The effect of β-lactam antibiotics viz. cefotaxime, carbenicillin and cephalexin was evaluated on shoot organogenesis of potato cultivars ‘Kufri Pukhraj’ and ‘Kufri Chipsona 1’. Leaf and internodal explants from 21-day-old microshoots were cultured on pre-standardized medium for shoot organogenesis (basal Murashige and Skoog (MS) medium containing 10 μM AgNO3+10 μM 6-benzyladenine (BA)+15 μM gibberellin A3 (GA3)), which was further supplemented with different concentrations (0–500 mg/L) of various β-lactam antibiotics. At lower concentrations, these antibiotics promoted shoot organogenesis in both cultivars, except cephalexin that totally inhibited shoot organogenesis in cv. ‘Kufri Chipsona 1’. Cefotaxime (100 mg/L) induced shoot organogenesis in maximum number of explants with maximum number of shoots per explant. Among explants, shoot promotion effect was significantly higher from internodal explants as compared with leaf explants. Cefotaxime at all concentrations (100–500 mg/L) increased shoot organogenesis of all explants and cultivars. The regenerated shoots tested clonally uniform among themselves and also to that of the mother plant by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers.