► Native carotenoids composition in 12 different cultivars of chilli peppers. ► HPLC-DAD-APCI-MS methodology. ► “Healthy” carotenoids were detected in the various cultivars investigated. ► Specific carotenoid profile in different cultivars of the same species. ► Pungency levels determined. In this research 12 different varieties of Capsicum cultivars belonging to three species (Capsicum chinense, Capsicum annuum, Capsicum frutescens) and of various colour, shape, and dimension have been characterised by their carotenoids and capsaicinoids content. The berries were cultivated in the region Emilia-Romagna, in Northern Italy. The native carotenoid composition was directly investigated by an HPLC-DAD-APCI-MS methodology, for the first time. In total, 52 carotenoids have been identified and considerable variation in carotenoid composition was observed among the various cultivars investigated. Among the cultivars with red colour, some Habanero, Naga morich and Sinpezon showed an high β-carotene content, whereas Serrano, Tabasco and Jalapeno showed an high capsanthin content and the absence of β-carotene. Habanero golden and Scotch Bonnet showed a high lutein, α-carotene and β-carotene amounts, and Habanero orange was rich in antheraxanthin, capsanthin and zeaxanthin. Cis-cryptocapsin was present in high amount in Habanero chocolate. The qualitative and quantitative determination of the capsaicinoids, alkaloids responsible for the pungency level, has also been estimated by a validated chromatographic procedure (HPLC-DAD) after a preliminary drying step and an opportune extraction procedure. Results have also been expressed in Scoville units. Dry matter and water activity have also been established on the fresh berries. The dried peppers of each variety were then submitted to the evaluation of the total nitrogen content, measured by a Dumas system, permitting to provide information on the protein content that was found to be in the range between 7 and 16%.