The aim of this study was to determine the pungency level of different accessions of Habanero peppers. The high-performance liquid chromatography (HPLC) technique was used to evaluate the content of total capsaicinoids in the whole fruit, placenta, and pericarp of 18 accessions of Habanero pepper from the germplasm bank of the Capsicum chinense species maintained in the Scientific Research Center of Yucatan [Centro de Investigación Científica de Yucatán (CICY)]. Thirteen of these accessions belonged to the “orange type”, four to the “red type”, and one to the “yellow type”. During the study, the plants were cultivated and maintained under greenhouse conditions and the fruit was harvested only when it was completely ripe on the plant. The results show considerable intraspecific diversity for this characteristic as well as the existence of cultivars of this species that surpass the levels of pungency reported for Habanero peppers under the conditions evaluated.