AbstractThe gonads of honey bee, Apis mellifera, queens and drones are each composed of hundreds of serial units, the ovarioles and testioles, while the ovaries of the adult subfertile workers consist of only few ovarioles. We performed a comparative RNA‐seq analysis on early fifth‐instar (L5F1) larval gonads, which is a critical stage in gonad development of honey bee larvae. A total of 1834 genes were identified as differentially expressed (Padj < 0.01) among the three sex and caste phenotypes. The Gene Ontology analysis showed significant enrichment for metabolism, protein or ion binding, and oxidoreductase activity, and a KEGG analysis revealed metabolic pathways as enriched. In a principal component analysis for the total transcriptomes and hierarchical clustering of the DEGs, we found higher similarity between the queen and worker ovary transcriptomes compared to the drone testis, despite the onset of programmed cell death in the worker ovaries. Four DEGs were selected for RT‐qPCR analyses, including their response to juvenile hormone (JH), which is a critical factor in the caste‐specific development of the ovaries. Among these, DMRT A2 and Hsp83 were found upregulated by JH and, thus, emerged as potential molecular markers for sex‐ and caste‐specific gonad development in honey bees.