CMX001 is an orally available lipid acyclic nucleotide phosphonate that delivers high intracellular levels of cidofovir (CDV)-diphosphate and exhibits enhanced in vitro antiviral activity against a wide range of double-stranded DNA viruses, including cytomegalovirus (CMV). Mutations in the DNA polymerase of CMV that impart resistance to CDV also render the virus resistant to CMX001. Here, we report a novel resistance mutation that arose under the selective pressure of CMX001. The wild-type CMV strain AD169 was propagated in human foreskin fibroblasts under increasing concentrations of CMX001 over 10 months, and the resulting strain (named CMX001(R)) was less susceptible to CDV and CMX001 in a plaque reduction assay. Genotypic analysis of virus strain CMX001(R) via conventional sequencing of the genes encoding the CMV DNA polymerase (UL54) and UL97 kinase (UL97) demonstrated one mutation that changed the wild-type aspartate to glutamate at position 542 in UL54. A recombinant virus with this novel D542E mutation was generated via bacterial artificial chromosome-mediated marker transfer experiments. Subsequent phenotypic resistance analysis of the D542E mutant demonstrated reductions in susceptibility of greater than 10-fold to CMX001 and CDV, but no resistance to foscarnet (FOS) or ganciclovir (GCV). Analysis of replicative fitness showed that both strain CMX001(R) and the D542E mutant viruses demonstrated a smaller plaque phenotype and slower replication kinetics than their respective parent viruses. These data describe the first resistance mutation generated under the selective pressure of CMX001 and suggest that CMX001 may have a unique resistance profile associated with reduced viral replication and maintenance of sensitivity to FOS and GCV.