In order to investigate the impairment of mitochondrial membrane phospholipid localization and DNA(3867) (mtDNA(3867)) deletion and the correlation between cardiac and skeletal muscle cells in mice with viral myocarditis, 50 BALB/c mice were divided into two groups randomly. In experimental group (n=40), the mice were intraperitoneally injected with 0.1 mL Eagle liquid with CVB3(TCID50=10(8)), while in the control group (n=10), the mice were subjected to equal volume of Eagle liquid. The impairment of mitochondrial membrane phospholipid localization and mtDNA(3867) deletion rate of cardiac and skeletal muscle were detected separately at day 3, 11 and 24 after injection. The correlation of mitochondrial membrane phospholipid localization and mtDNA(3867) deletion rate between cardiac and skeletal muscle cells cells was analyzed using Spearman method. At the day 3 after injection, in both cardiac and skeletal muscle cells, mtDNA(3867) deletion rate was significantly higher in experimental group than in control group (P<0.05), but the localization of mitochondrial membrane phospholipid showed no difference between two groups (P>0.05). At day 11 after injection, the mtDNA(3867) deletion rate of both cells in experimental group was increased to the peak level (P<0.05), and the impairment of mitochondrial membrane phospholipid localization of both cells also increased markedly in experimental group as compared with control group (P>0.05). At the day 24 after injection, the impairment of mitochondrial membrane phospholipid localization and mtDNA(3867) deletion of both cells showed a recovery tendency, but still severer than those at the day 3 after injection (P<0.05). The impairment of mitochondrial membrane phospholipid localization and mtDNA(3867) deletion were consistent and synchronistic between cardiac and skeletal muscle cells, and showed good correlations (P<0.05). The impairment of mitochondria plays an important role in the pathogenesis of viral myocarditis, and the skeletal muscle cells might act as a peripheral "window" to reflect the mitochondrial damage of cardiac myocytes.