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• Capsicum annuum (Long pepper) × Capsicum frutescens (PI281420) were selfed. • Total phenolics, flavonoid and total anthocyanin contents of the red pepper lines changed significantly. • Scoville heat unit revealed that the 95% of the peppers were highly pungent. • The major phenolics of peppers were detected as p-coumaric acid. In this study, 104 RIL (Recombinant Inbred Pepper Lines: F6) populations which generated by selfing Capsicum annuum (Long pepper) × Capsicum frutescens (PI281420) F6 population were characterized in terms of detailed bioactive properties, major phenolic composition, tocopherol and capsaicinoid profile. Total phenolics, flavonoid and total anthocyanin contents of the red pepper lines were in the range of 7.06–17.15 mg gallic acid equivalent (GAE)/g dw, 1.10–5.46 mg catechin equivalent (CE)/g dw and 7.9–516.6 mg/kg dw extract, respectively. Antiradical activity and antioxidant capacity values also ranged between 18.99 and 49.73% and 6.97–16.47 mg ascorbic acid equivalent (AAE)/kg dw, respectively. Capsaicin and dihydrocapsaicin levels showed a wide variance with the range of 27.9–1405.9 and 12.3–640.4 mg/100 g dw, respectively. Scoville heat unit revealed that the 95% of the peppers were highly pungent. The major tocopherol was alpha tocopherol for the pepper samples with the highest level of 1078.4 µg/g dw. The major phenolics were detected as p-coumaric acid, ferulic acid, myricetin, luteolin and quercetin. Pepper genotypes showed significant differences in terms of the characterized properties and principal component analysis was applied successfully to reveal the similar genotypes. [ABSTRACT FROM AUTHOR]