Heading date of rice is a key agronomic trait determining cultivated areas and seasons and affecting yield. In the present study, five primary single segment substitution lines with the same genetic background were used to detect quantitative trait loci (QTLs) for heading date in rice. Two QTLs, qHD3 and qHD6 on the short arm of chromosome 3 and the short arm of chromosome 6, respectively, were identified under natural long-day (NLD). Nineteen secondary single segment substitution lines (SSSLs) and seven double segments pyramiding lines were designed to map the two QTLs and to evaluate their epistatic interaction between them. By overlapping mapping, qHD3 was mapped in a 791-kb interval between SSR markers RM3894 and RM569 and qHD6 in a 1 125-kb interval between RM587 and RM225. Results revealed the existence of epistatic interaction between qHD3 and qHD6 under natural long-day (NLD). It was also found that qHD3 and qHD6 had significant effects on plant height and yield traits, indicating that both of the QTLs have pleiotropic effects.