Borrelia burgdorferi, the etiological agent of Lyme disease in humans, is vectored between mammalian hosts in nature by Ixodes ticks. The organism adapts to diverse environments encountered throughout the enzootic cycle by differentially expressing essential gene products to survive the specialized conditions, whether in ticks or warm-blooded hosts. However, little is known regarding the identity and/or function of B. burgdorferi genes expressed during colonization of tissues during mammalian infection. Experimental evidence has shown that a group of genes (formerly classified as paralogous gene family 54) contiguously localized on the 54-kilobase linear plasmid of B. burgdorferi, are among the most highly regulated by in vitro conditions resembling mammalian infection. In this study, we employed quantitative reverse transcription-PCR to measure temporal gene expression of a subset of this B. burgdorferi gene family (bba64, bba65, bba66, and bba73) in tissues during chronic murine infection. The goal was to gain insight into the role of these genes in infectivity and pathogenesis by identifying when the genes are induced and whether they are expressed in specific target tissues. B. burgdorferi bba64, bba65, bba66, and bba73 expression was measured from infected mouse tissues relative to expression in in vitro culture conditions at specific times post-infection. bba64 expression was highly upregulated in bladder, heart, and spleen tissues throughout the infection period, contrasting with the sharp downregulation previously observed in ear tissues. bba65, bba66, and bba73 demonstrated upregulated differential expression in various tissues over 1 year post-infection. These results suggest an essential role for these genes in borrelial survival, persistence, and/or pathogenesis.