Cytoplasmic effects are important agronomical phenomena that have generated widespread interest in both theory and application. In the present study, five high yield rice cultivars(Oryza sativa L. ssp. japonica) in large-scale cultivation in northeast China were determined to possess Oryza sativa L. ssp. indica-type cytoplasm using cytoplasmic subspecies-specific molecular markers. This was confirmed by cytoplasmic genome-wide single nucleotide polymorphisms(SNPs) and functional gene sequencing. Two of these five japonica cultivars were core breeding parents with high yield and the other three were super-high-yield varieties registered by the Ministry of Agriculture of China. We constructed nuclear substitution lines to further demonstrate whether and how this indica-type cytoplasm contributed to yield improvement by comparing yield components. The results showed that under the same japonica nuclear background, the lines with indica-type cytoplasm had a significant decrease in tillers in exchange for increased grain number per panicle compared with their recurrent parents. Our results implied that botanical basis of this cytoplasmic effect was to reduce the plant’s branching differentiation to produce more floral organs under the constant nutrition. Our findings open another door for the utilization of inter-subspecific hybridization for the improvement of rice cultivar. Cytoplasmic effects are important agronomical phenomena that have generated widespread interest in both theory and application. In the present study, five high yield rice cultivars (Oryza sativa L. ssp. Japonica) in large-scale cultivation in northeast China were determined to possess Oryza sativa L. wasp. Indica-type cytoplasm using cytoplasmic subspecies-specific molecular markers. This was confirmed by cytoplasmic genome-wide single nucleotide polymorphisms (SNPs) and functional gene sequencing. Two of these five japonica cultivars were core breeding parents with high yield and the other three were super-high-yield varieties registered by the Ministry of Agriculture of China. We constructed nuclear substitution lines to further demonstrated whether and how this indica-type cytoplasm contributed to yield improvement by comparing yield components. The results showed that under the same japonica nuclear background, the lines with indica-type cytoplasm had a significant decrease in tillers in exchange for increased grain number per panicle compared with their recurrent parents. Our results implied that botanical basis of this cytoplasmic effect was to reduce the plant’s branching differentiation to produce more floral organs under the constant nutrition. inter-subspecific hybridization for the improvement of rice cultivar.