Background: Heterotopic ossification frequently develops following high-energy blast injuries sustained in modern warfare. We hypothesized that differences in the population of progenitor cells present in a wound would correlate with the subsequent formation of heterotopic ossification. Methods: We obtained muscle biopsy specimens from military service members who had sustained high-energy wartime injuries and from patients undergoing harvest of a hamstring tendon autograft. Plastic-adherent cells were isolated in single-cell suspension and plated to assess the prevalence of colony-forming cells. Phenotypic characteristics were assessed with use of flow cytometry. Individual colony-forming units were counted after an incubation period of seven to ten days, and replicate cultures were incubated in lineage-specific induction media. Immunohistochemical staining was then performed to determine the percentage of colonies that had differentiated along an osteogenic lineage. Quantitative real-time reverse-transcription polymerase chain reaction was used to identify changes in osteogenic gene expression. Results: Injured patients had significantly higher numbers of muscle-derived connective-tissue progenitor cells per gram of tissue (p < 0.0001; 95% confidence interval [CI], 129,930 to 253,333), and those who developed heterotopic ossification had higher numbers of assayable osteogenic colonies (p < 0.016; 95% CI, 12,249 to 106,065). In the injured group, quantitative real-time reverse-transcription polymerase chain reaction performed on the in vitro expanded progeny of connective-tissue progenitors demonstrated upregulation of COL10A1, COL4A3, COMP, FGFR2, FLT1, IGF2, ITGAM, MMP9, PHEX, SCARB1, SOX9, and VEGFA in the patients with heterotopic ossification as compared with those without heterotopic ossification.