Abstract: A total of 118,900 ESTs in Capsicum annuum L were downloaded from NCBI for identifying and developing an SSR marker. As a result, 3,343 SSRs in the ESTs with an average of one SSR per 17.20 kb were found, including 283 SSR motifs. Analysis on the SSR motifs revealed that they were mostly di-nucleotides (1,476 SSR) and hexa-nucleotides (1,128 SSR), accounting for 44.15% and 33.74% of the total, respectively. GA/TC was the most frequently found motifs (14.30% of all), which was followed by CT/AG (94 SSR, and 8.11% of all). From the 3,343 SSR-containing ESTs, 200 primer pairs were designed and validated for amplification using 17 pepper inbred lines. The results showed that 71 primer pairs yielded 220 amplification bands. Using those 71 pairs of polymorphic primers, the genetic relationship between the parents, P181-2-6 and P230-2-5, and their F1 hybrid was determined. It appeared that the SSR markers identified from the ESTs of C. annuum were potentially of practical applicatios.