Aims: The aim of this paper is to develop an in vitro organogenesis and callogenesis protocol for Carolina Reaper pepper, and to determine the karyotype and nucleoli of this cultivar. Methodology: The MS medium with supplemented with indole-3-butyric (0, 1, 2 and 4 mg L-1) and kinetin (0, 1, 2 and 4 mg L-1) was used. The leaves and nodal segments of Carolina reaper was utilized for the callogenesis and organogenesis induction. The responses to growth regulators were evaluated 30 days of cultivation. The meristematic tissue was pre-treated with 0.05% (w/v) of colchicine for six hours at 18°C. The samples were fixed in Carnoy for 12 hours. Chromosomal observations were made with binocular optical microscope (Leica DM 750) and the cells in good condition for counting the chromosomes and karyotype assembly were photographed. Results were presented as mean ± standard deviation and were compared by the two-way Analysis of Variance. The means were separated according to Tukey test (P = 0.05). Results: Calli were induced from both leaf and stem segments when indole-3-butyric 0 mg L-1 + kinetin 1 mg L-1 were used. Development of shoots in leaf and stem segment were obtained when indole-3-butyric 2 mg L-1 + kinetin 4 mg L-1 were used, and roots regenerated with indole-3-butyric 4 mg L-1 + kinetin 1 mg L-1. It was found two nucleoli in every cell interphase, suggesting that two nucleolar organizer regions are expressing their ribosomal genes. Karyotype analysis indicated a chromosome number of 2n = 24, which is correlation with other Capsicum genus varieties. It was observed 1 or 2 nucleoli per nucleus of both types, homomorphic and heteromorphic. The results can help in programs of breeding and conservation of this cultivar and other species of pepper. Conclusion: Using the concentrations of growth hormones indicated in the present report, it could be possible to regenerate leaves and nodal segments in vitro clones from the original genotype. We have also described the chromosome number and nucleolus number of Carolina reaper, generating a data that could help in programs of breeding, as in the generation of polyploid plants and conservation species of pepper.