A stoma is a pore in the leaf or stem epidermis for exchanging CO2 and water vapor that plays a significant role in photosynthesis. To improve photosynthetic ability, many biological studies focusing on stomata development have been performed. However, since the characterization of stomata shape and distribution requires time and effort, the genetic basis of stomata development in rice is still unclear. We focused on stomatal density as an index of photosynthetic ability, and developed semi-automated software for counting the number of stomata on scanning electron microscope image of abaxial leaf epidermis. High-yielding indica rice cultivar, Takanari, has a higher stomata density on the flag leaf than japonica rice cultivar, Koshihikari. Quantitative trait loci (QTL) analysis using backcross inbred lines derived from a cross between Koshihikari and Takanari revealed that there are three QTLs involving this phenotypic variation. The most prominent QTL was located on the long arm of chromosome 6. To confirm the effect of this QTL, we surveyed stomata density in a series of chromosomal segment substitution lines (CSSLs), in which a part of genome segment is replaced with Takanari under Koshihikari genetic background. This allowed the QTL to be mapped to within about 3.3 Mbp. Furthermore, CSSLs with the corresponding segments from Takanari showed increased leaf photosynthetic rate and increased stomata density compared to Koshihikari. This result implies that the detected QTL involving stomatal density affects the leaf photosynthesis rate.