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Interspecific hybridization is intensively carried out to increase the existing genetic variation and to extend of genetic bases of varieties that have biotic, abiotic stresses tolerance and also genetic studies to determine variation of nutritional characteristics in pepper breeding programs. In this study, 104 RIL (Recombinant Inbred Pepper Lines: F6) populations which generated by selfing C. annuum (Long pepper) x C. frutescens (PI281420) F6 population were used to assessment of some fruit nutritional traits. Crude oil ratios varied between 3.20% and 11.43%, crude ash ratios between 5.26% and 17.69% and crude protein ratios between 8.09% and 23.57%. Na contents varied between 106.04 and 2140.07 ppm, Ca contents between 1167.67 and 6004.13 ppm, K contents between 10419.03 and 24775.40 ppm, Mg contents between 1240.37 and 6793.53 ppm, P contents between 2125.23 and 8858.85 ppm and S contents between 1037.65 and 7691.55. Lauric acid contents varied between 0.03% and 0.92%, myristic acid contents between 0.10% and 2.93%, palmitic acid contents between 11.67% and 27.13%, palmitoleic acid contents between 0.18% and 1.67%, heptadecanoic acid contents between 0.00% and 0.53%, heptadecenoic acid contents between 0.00% and 0.25%, stearic acid contents between 1.57% and 6.79%, oleic acid contents between 7.01% and 49.98%, linoleic acid contents between 26.28%− 57.79%, arachidic acid contents between 0.12% and 1.92%, linolenic acid contents between 0.97% and 14.86%, behenic acid contents between 0.05% and 2.58%, MUFA contents between 8.58% and 51.12% and PUFA contents between 28.98% and 68.51%. Beta-carotene contents varied between 23.92 and 1547.19 mg/100 g. Interspecific hybridization in pepper has an important role in increased the nutritional properties level in hybrids. So, these results open the new world gene pool for pepper breeding, ultimately contributing to nutritional properties to new pepper varieties. • Wide variation was achieved by interspecies crossing. • Some inbred lines have better nutritional properties than parents. • Protein content, PUFA, S, P, Mn, Mg were determined as ideal properties in lines. • Variation of nutritional properties can be used for mapping of related genes and breeding. [ABSTRACT FROM AUTHOR]