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Throughout the globe morphological, biochemical and genetic variability exists in chilli and is harnessed to achieve specific breeding objectives. In this study, chilli germplasm was characterized based on horticultural traits, biochemical quantification and simple sequence repeat (SSR) polymorphism for diversity estimation. A total of 36 SSR primers were utilised to study the genetic divergence among 48 genotypes of chilli collected from nine states of India. Among the 36 primers, sixteen amplified null alleles. A total of 41 alleles were detected with average 2.05 alleles per locus. The largest number of alleles (5) were obtained with marker CAMS-234. The polymorphic information content ranged from 0.06 to 0.72 with an average of 0.50. On the basis of SSR analysis, the UPGMA cluster classified 48 genotypes into three groups. There was significant variability in germplasm for all morpho-biochemical traits. Kashi Anmol (100.50 q/ha) expressed the highest yield. Highest vitamin C content at green stage was recorded in IC-561635 (187 mg/100 g) and the greatest capsaicin content (9547.90 µg/g) equivalent to pungency of 171,862.2 Scoville heat units (SHU) was recorded in Bhut Jolokia. Principal component analysis indicates that the first five principal components explain 74.63% per cent of the total variation. Additionally, analysis of molecular variance (AMOVA) showed that 1% of the total genetic variation occurred among the population and 99% genetic variation within the populations, whereas the pairwise Fst specified the moderate genetic variation ranging from 0.002 to 0.020. The present investigation has strengthened the knowledge of genetic worth of this germplasm for application in various genetic improvement programmes. [ABSTRACT FROM AUTHOR]