The human body consists of innumerable multifaceted environments that predispose colonization by a number of distinct microbial communities, which play fundamental roles in human health and disease. In addition to community surveys and shotgun metagenomes that seek to explore the composition and diversity of these microbiomes, there are significant efforts to sequence reference microbial genomes from many body sites of healthy adults. To illustrate the utility of reference genomes when studying more complex metagenomes, we present a reference-based analysis of sequence reads generated from 55 shotgun metagenomes, selected from 5 major body sites, including 16 sub-sites. Interestingly, between 13% and 92% (62.3% average) of these shotgun reads were aligned to a then-complete list of 2780 reference genomes, including 1583 references for the human microbiome. However, no reference genome was universally found in all body sites. For any given metagenome, the body site-specific reference genomes, derived from the same body site as the sample, accounted for an average of 58.8% of the mapped reads. While different body sites did differ in abundant genera, proximal or symmetrical body sites were found to be most similar to one another. The extent of variation observed, both between individuals sampled within the same microenvironment, or at the same site within the same individual over time, calls into question comparative studies across individuals even if sampled at the same body site. This study illustrates the high utility of reference genomes and the need for further site-specific reference microbial genome sequencing, even within the already well-sampled human microbiome.