Capsaicinoids, a group of secondary metabolites (alkaloids) which imparts hot property, are unique to the Capsicum fruits and have high value in the pharmaceutical and food industries. In spite of having high capsaicinoids content with ethnopharmacological and economic importance, the C. chinense and C. frutescens of the Northeast India, are underexplored for the genetics and breeding programs. In this study, we developed transcriptome-based Simple sequence repeat (SSR) markers and analyzed the expression(s) of capsaicinoids and carotenoid biosynthesis pathway genes in flower and fruit tissues at three developmental stages (early, breaker and mature) of C. chinense, C. frutescens and C. annuum. A total of 4988 and 4781 genic SSRs were identified from 123,118 and 121,017 de novo assembled unigenes, in C. chinense and C. frutescens, respectively. Also, the reference-based transcriptome analysis identified 70.8–73.6 % commonly expressed transcripts in all tissues besides 2929, 1327, 1193 and 937 unique transcripts in flower, early, breaker and mature fruits, respectively. The gene expression profiling showed significantly high expression of the key capsaicinoids and carotenoid biosynthetic pathway genes at breaker and mature fruit stages. Furthermore, in silico analysis identified a total of 335 polymorphic SSRs between C. chinense and C. frutescens with allelic size difference of >4 bases. The trinucleotide repeats was found to be predominant in both C. chinense (50.6 %) and C. frutescens (52.2 %). High genetic-diversity of SSRs was observed with mean gene diversity of 0.51 and polymorphism information content (PIC) of 0.53. Based on twenty polymorphic SSRs, the UPGMA cluster analysis differentiated 96 genotypes belonging to C. chinense, C. frutescens and C. annuum into distinct groups. The identified SSRs and diverse Capsicum genotypes will serve as important genetic resources for future applications in genetics study and breeding of Capsicum varieties with improved metabolites (pungency, carotenoids etc.) and agronomic traits (fruit shape, size etc.) for agricultural, food and pharmaceutical industries.