In this study, 137 629 transcripts were assembled from 8.9 Gb of clean Illumina DNA sequencing read data, yielding 83 811 unigene sequences from Pueraria thomsonii ‘No. 1’. A phylogenetic analysis indicated that Pueraria lobata and Medicago sativa clustered together with Arachis hypogaea (peanut). We detected 25 452 SSR loci in the 83 811 assembled unigenes using MISA software. Trinucleotide repeats were the most abundant followed by dinucleotide repeats. Among the trinucleotide motifs, (AAG)_n (27.87%), was the most common repeat unit. A total of 229 SSR primer pairs were designed, and 28 markers that gave clear, polymorphic amplification products were used to analyze the genetic diversity within a panel of 44 Pueraria accessions. Ninety SSR fragments, consisting of 89 alleles, were amplified from genomic DNA of the 44 accessions. The average allele number is 3.178 6. Polymorphic information content (PIC) values ranged between 0.083 0 and 0.774 2 (mean = 0.455 7). Cluster analysis showed that the genetic similarity coefficients among the accessions ranged from 0.266 7 to 1.000 0. These results suggest that the detected P. lobata resources have abundant genetic diversity at DNA molecular level. The ingroup similarity coefficient (0.58) was observed in the 44 germplasm accessions, and all accessions could be clearly divided into two groups. The clustering results of tested P. lobata resource did not show clear correlation to their geographic origin. These markers are reliable genomic resource for genetic diversity analysis in Pueraria.