Prickly lettuce (Lactuca serriola L.) is a problematic weed of Pacific Northwest and recently developed resistance to the auxinic herbicide 2,4-D. There are no publically available simple sequence repeat (SSR) markers to tag 2,4-D resistance genes in L. serriola. Therefore, a study was conducted to develop SSR markers from expressed sequence tags (ESTs) of 5 Lactuca species. A total of 15,970 SSRs were identified among 57,126 EST assemblies belonging to 5 Lactuca species. SSR-containing ESTs (SSR-ESTs) ranged from 6.23% to 7.87%, and SSR densities ranged from 1.28 to 2.51 kb(-1) among the ESTs of 5 Lactuca species. Trinucleotide repeats were the most abundant SSRs detected during the study. As a representative sample, 45 ESTs carrying class I SSRs (≥ 20 nucleotides) were selected for designing primers and were also searched against the dbEST entries for L. sativa and Helianthus annuus (≤ 10(-50); score ≥ 100). In silico analysis of 45 SSR-ESTs showed 82% conservation across species and 68% conservation across genera. Primer pairs synthesized for the above 45 EST-SSRs were used to study genetic diversity among a collection of 22 L. serriola biotypes. Comparison of the resultant dendrogram to that developed using phenotypic evaluation of the same subset of lines showed limited correspondence. Taken together, this study reported a collection of useful SSR markers for L. serriola, confirmed transferability of these markers within and across genera, and demonstrated their usefulness in studying genetic diversity.