Mitochondrial DNA (mtDNA) is uniparentally inherited in most sexual eukaryotes called maternal/uniparental inheritance. It is well known that selective elimination of paternal mitochondria and digestion of paternal mtDNA are important to this process. However, little is known about the control of maternal mitochondrial proliferation. In the isogamous Physarum polycephalum, haploid myxamoebae act as gametes. When two myxamoebal strains with different mating types are mated, mtDNA is inherited from only one strain, defined as the maternal strain. Here, we developed a method to distinguish uniparental mitochondria in zygotes of P. polycephalum by chemical staining and investigated the proliferation of maternal and paternal mitochondria during the selective digestion of mitochondrial nucleoids (mt-nucleoids) in paternal mitochondria. We stained the myxamoebae with MitoTracker Red CMXRos (MTR) or MitoTracker Green FM (MTG). MTG-stained mitochondria more specifically than MTR. When the AI35 strain was crossed with the MTG-stained DP246 strain and mtDNA was stained with N-aryl pyrido cyanine 3, mt-nucleoids in mitochondria derived from DP246 became shorter in the zygotes 3 h after crossing. There was no significant difference in the number of mitochondria derived from both AI35 and DP246 in early zygotes, but the number of mitochondria derived from AI35 proliferated 1.7-fold from 3 to 5 h after crossing. Similar results were obtained when MTG-stained AI35 crossed with DP246. These results indicate that the proliferation of mitochondria derived from AI35 rapidly occurred after the initiation of digestion of mt-nucleoids derived from DP246. Our results suggest the possibility of controlling the mechanism of the selective proliferation of maternal mitochondria during zygote formation for uniparental inheritance.