A total of 32 295 non-redundant expressed sequence tags (ESTs) in pepper were screened by using bioinformatics software to search for SSR motifs, 3 396 SSRs were sought out, distributing in 3 277 ESTs, corresponding to one SSR in every 4.46 kb of the ESTs. Dinucleotide and trinucleotide repeats were major types among the obtained unigenes, accounting for 43.02% and 37.84%, respectively. GA/TC (15.99%),AG/CT(11.98%) and AT(11.37%) were the most abundant motifs. Based on the flanking sequences of these 3 396 SSRs, 420 primer pairs were designed by using Primer Premier 5.0 software. 403 SSRs (95.96%) were successfully amplified and 76 of them (18.86%) showed polymorphism between ‘B072’ (the resistance inbred line) and ‘B088’ (the susceptible inbred line). These results proved that it is an effective and feasible approach to developing SSR markers based on ESTs in Capsicum annuum L.