Duchenne muscular dystrophy (DMD) is a rare, fatal X-linked disorder characterized by the lack of dystrophin, a key sarcolemma muscle protein. Cardiac failure is a significant cause of death in DMD subjects. The purpose of our research was to identify potential cardiac serum biomarkers associated with DMD cardiomyopathy. This is an observational, case-controlled study using subjects from the CINRG DMD natural history study with cardiomyopathy (ejection fraction (EF) <55%; shortening fraction (SF) <28%), subjects without cardiomyopathy (EF ≥ 55%; SF ≥ 28%) compared to normal healthy volunteer subjects. The DMD with cardiomyopathy group had significantly lower average EF and SF (EF = 45 ± 10/SF = 25 ± 2%) than the DMD without cardiomyopathy group (EF = 58 ± 5% and SF = 32 ± 3%; p < 0.01). Among a selected set of potential biomarkers for cardiomyopathy (MMP9, BNP, GAL3, CRP, LEP, TNC, TLR4 and ST2) we validated ST2 as significantly elevated in the serum of DMD cardiomyopathy group (35,798 ± 4884 pg/mL) compared to normal controls (9940 ± 2680 pg/mL; p < 0.01; n = 6). Matrix metallopeptidase 9 (MMP9) levels were found significantly increased in both DMD groups compared to controls (p < 0.01). No significant differences were seen in BNP, GAL3, CRP, LEP, TNC or TLR4 levels. Increased ST2 levels were found in serum of DMD subjects compared to healthy volunteers and further elevated in DMD subjects with cardiomyopathy. Future studies correlating cardiomyopathy with ST2 levels may allow for improved non-invasive monitoring of cardiac disease in DMD subjects.