Cytoplasmic male sterility (CMS) has often been associated with mitochondrial dysfunction. In this report, the heterogeneity of mitochondria was analyzed in both Honglian (HL) CMS (YtA) rice seedlings and those of its corresponding maintainers (YtB) by flow cytometry and staining with rhodamine-123 (Rh-123). Both lines revealed two distinct fluorescence populations: high fluorescence populations (HFP) and light fluorescence populations (LFP), and a somewhat lower LFP/HFP ratio was detected in conjunction with the higher reactive oxygen species (ROS) content in YtA. In addition, use of the specific effector hydrogen peroxide (H2O2) demonstrated a correlation between the LFP/HFP ratio and ROS levels in both lines. Higher ROS content caused a more swift decrease of F(0)F(1)-ATPase activity and ATP contents in YtA than those in YtB, which accompanied with an obvious decline of the LFP/HFP ratio in YtA. Furthermore, a mitochondrial genomic DNA smear was detected by pulsed field gel electrophoresis. Taken together, these results implied that HL-CMS line rice seedlings and those of its corresponding maintainer have different proportion of Rh-123 staining mitochondria populations, which may be accounted for by ROS contents on the basis of ATPase activity and ATP contents.