Rocoto (Capsicum pubescens Ruiz & Pav.) is a native plant of Peru, used in the national gastronomy with great nutritional value; it has pharmaceutical and medicinal properties. The crop is susceptible to diseases caused by phytopathogens, which are spread by seeds of infected cultivars or seedlings. Through the use of biotechnological tools such as in vitro plant tissue culture, it is possible to obtain plants free of pathogens, of good quality and great agronomic potential. Therefore, the present work aimed to develop a methodology for the in vitro induction of callus from hot pepper leaves. In vitro germinated rocoto seedlings were used, from which the first true leaves were selected, which were sectioned into 1 cm explants and placed in different MS culture media added with 2,4-dichlorophenoxyacetic acid (2,4-D). The induction response to calllogenesis was evaluated in five treatments with different concentrations of 2,4-D (0, 0.25, 0.5, 0.75 and 1 mg l−1), under conditions of 25 ◦C in the dark for 35 days. The culture media added with 0.75 and 1 mg l−1 of 2.4-D allowed to obtain 100% induction of calluses in the hot pepper leaves with 81% and 86%, respectively of grade 3 callus formation. This study, pioneer for the species, is good for the potential use of breeding programs.