The genetic diversity among 23 newly developed homosegregate pea lines (Pisum sativum L.) was assessed with a total of 13 expressed sequence tag (EST) based-simple sequence repeat (SSR) markers. The percentages of amplified and nonamplified primers were 92% and 8%, respectively, and 58.33% of the used primers gave the PCR product within the reported size range while 41.66% of primers gave a different product size. Polymorphism information content (PIC), major allele frequency, and variation in genetic diversity were calculated. The PIC ranged from 0.32 to 0.63 with an average of 0.50. Major allele frequency ranged from 0.48 to 0.78 with a mean value of 0.56. The variation in genetic diversity among these pea lines ranged from 0.36 to 0.68 with a mean value of 0.56. Cluster analysis based on a dendrogram divided the 23 pea lines into two main groups (L-1 and L-2), separated at 25% genetic distance. Seven subclusters were evident from these two main groups. L-1 grouped 51.2% (12 pea lines) while L-2 contained 47.8% (11 pea lines) of the total analyzed population. It was concluded that EST-SSR markers are useful for refinement of the pea linkage map.