Pepper (Capsicum annuum L.) is one of the staple vegetable crops with significant economic value. Investigation of pepper genetic diversity is crucial for the collection, identification and application of pepper germplasm resources. EST-SSR markers, derived from the transcript regions, can detect the polymorphism in the transcript regions on the genome and thus exhibit the ‘true genetic diversity’. In the study, a total of 33 EST-SSR markers were employed to investigate the genetic diversity harbored among 31 pepper varieties. A total of 91 alleles were detected among the tested pepper varieties by the EST-SSR markers. The maximum number of alleles and the average alleles per locus amplified by EST-SSR markers were 6 and 2.76 respectively. The polymorphic information content (PIC) per locus ranged from 0.03 to 0.74 with the average of 0.38. Systemic cluster analysis separated the tested pepper varieties into two major groups which showed certain correlation with the morphologic traits of the fruit. Principal coordinate analysis acquired the similar results with the systemic cluster analysis, but exhibiting more thoroughly genetic variations harbored among the tested pepper varieties.