We used field trials to study the potential non-target effects of transgenic chili pepper on the composition of a rhizosphere microbial community. These plants expressed a viral coat protein (CP) gene that confers resistance to cucumber mosaic virus (CMV). For comparison, we included a non-transgenic parental cultivar (wild type) and another non-transgenic commercial variety. Soil samples were collected at the time of flowering (June) and twice during the fruit-ripening stage (August and September). The microbial community was characterized by analyzing levels of phospholipid fatty acids (PLFAs) as well as the terminal restriction fragment length polymorphism (T-RFLP) of bacterial 16S-rRNA and the fungal internal transcribed spacer (ITS) region. Although the amounts of PLFAs and their diversity indices did not differ among pepper lines, they did vary significantly over the growing season. We also used non-metric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PerMANOVA) to evaluate microbial assemblages from these plants. Overall, no differences were found among pepper lines. However, a significant difference was found between wild type and transgenic peppers, with regard to their bacterial composition based on the T-RFLP profile from HhaI digest. We conclude that the impact of CMV-resistant transgenic pepper on soil microbial assemblages is weak and minor, compared with the dominating natural variations associated with plant growth stages.