Wheat-Haynaldia villosa chromosome substitution line (6A/6V) and translocation lines (6DL/6VS, 6AL/6VS) were obtained through hybridization of H. villosa with powdery mildew susceptible cultivated wheat. Substitution line and translocation lines contain V chromosome or the chromosome short arm (VS) of H. villosa. They are resistant to powdery mildew. In this study, mitochondrial proteome changes were analyzed by using substitution line (6A/6V), translocation line (6DL/6VS) as experimental materials in order to studying the effects of V chromosome on the mitochondrial proteome and related to powdery mildew resistance. The results indicated that 16 new mitochondrial protein spots (spot1, 22kDa/PI8.5; spot2, 31 kDa/PI 7.5; spot3, 28 kDa/PI 7.0; spot4, 31 kDa/PI 6.5; spot5, 40 kDa/PI 7.5; spot6, 40 kDa/PI 7.4; spot7, 80 kDa/PI 8.4; spot8, 50 kDa/PI 7.5; spot9, 60 kDa/PI 7.3; spot10, 65 kDa/PI 6.6; spot11, 65 kDa/PI 6.6; spot12, 73 kDa/PI 7.5; spot13, 73 kDa/PI 7.7; spot14, 46 kDa/PI 7.4; spot15, 46 kDa/PI 7.3; spot16, 38 kDa/PI 6.3) were produced and 7 mitochondrial protein spots (spot1, 40 kDa/PI 7.5; spot2, 43 kDa/PI 7.6; spot3, 48 kDa/PI 7.5; spot4, 42 kDa/PI 8.0; spot5, 43 kDa/PI 7.5; spot6, 32 kDa/PI 4.8; spot7, 40 kDa/PI 5.5) were absent in substitution line, 7 new mitochondrial protein spots (spotl, 43 kDa/PI 6.3; spot2, 60 kDa/PI 6.5; spot3, 60 kDa/PI 6.4; spot4, 65 kDa/PI 7.5; spot5, 55 kDa/PI 8.2; spot6, 31 kDa/PI 8.0; spot7, 43 kDa/PI 8.0) were produced and 6 mitochondrial protein spots (spot1', 66 kDa/PI 8.3; spot2', 58 kDa/PI 8.5; spot3', 36 kDa/PI 7.0; spot4', 48 kDa/PI 7.7; spot5', 48 kDa/PI 6.8; spot6', 43 kDa/PI 6.2) were absent in translocation line. These experimental results suggest that V chromosome or VS of H. villosa can obviously lead mitochondrial proteome changed. These changes may be associated with resistant to powdery mildew of substitution line and translocation line.