Ixodes scapularis ticks play an important role in the transmission of a wide variety of pathogens between various mammalian species, including humans. Pathogens transmitted by ticks include Borrelia, Anaplasma and Babesia. Although ticks may harbour both pathogenic and non-pathogenic microflora, little is known about how the diversity of the microflora within ticks may influence the transmission of pathogens. To begin addressing this question, we examined the composition of bacterial communities present in Ixodes scapularis collected from Westchester and Dutchess Counties, New York State, at different developmental and nutritional stages. Genetic fingerprints of bacterial populations were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis for bacterial identification. The fingerprints of the TTGE bands were grouped into five clusters. The most abundant DNA sequence found in all the samples was Rickettsia, followed by Pseudomonas and Borrelia. Ralstonia, Anaplasma, Enterobacterias, Moraxella, Rhodococcus and uncultured proteobacterium were present as well. We also determined the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi by PCR and DNA sequence analysis. Statistical analyses indicated significant variations in the bacterial communities depending on tick developmental stage and degree of engorgement. We suggest that these two elements affect microbial diversity within the tick and may in turn influence pathogen transmission to humans and animals after tick bite.