To evaluate caste-biased gene expression in Melipona quadrifasciata, a stingless bee, we generated 1278 ESTs using Representational Difference Analysis. Most annotated sequences were similar to honey bee genes of unknown function. Only few queen-biased sequences had their putative function assigned by sequence comparison, contrasting with the worker-biased ESTs. The expression of six annotated genes connected to caste specificity was validated by real time PCR. Interestingly, queens that were developmentally induced by treatment with a juvenile hormone analogue displayed an expression profile clearly different from natural queens for this set of genes. In summary, this study represents an important first step in applying a comparative genomic approach to queen/worker polyphenism in the bee.