Abstract Background Increasing evidence demonstrates that stem cells maintain their identities by a unique transcription network and chromatin structure. Opposing epigenetic modifications H3K27me3 and H3K4me3 have been proposed to label differentiation-associated genes in stem cells, progenitor and precursor cells. In addition, many differentiation-associated genes are maintained at a poised status by recruitment of the initiative RNA Polymerase II (Pol II) at their promoter regions, in preparation for lineage-specific expression upon differentiation. Previous studies have been performed using cultured mammalian embryonic stem cells. To a lesser extent, chromatin structure has been delineated in other model organisms, such as Drosophila, to open new avenues for genetic analyses. Results Here we use testes isolated from a Drosophila bag of marbles mutant strain, from which germ cells are in their undifferentiated status. We use these testes to study the endogenous chromatin structure of undifferentiated cells using ChIP-seq. We integrate the ChIP-seq with RNA-seq data, which measures the digital transcriptome. Our genome-wide analyses indicate that most differentiation-associated genes in undifferentiated cells lack an active chromatin mark and initiative Pol II; instead, they are associated with either the repressive H3K27me3 mark or no detectable mark. Conclusions Our results reveal that most of the differentiation-associated genes in undifferentiated-cell-enriched Drosophila testes are associated with monovalent but not bivalent modifications, a chromatin signature that is distinct from the data reported in mammalian stem or precursor cells, which may reflect cell type specificity, species specificity, or both.