To examine whether parental alleles were randomly transmitted from heterozygous donor plants to anther culture (AC)-derived plants, two AC-derived populations and one F-2 population in rice were compared for 50 RFLP markers distributed on the rice chromosomes. Two populations which were developed through different anther culture methods (the ordinary method and the direct regeneration method) and the F-2 population were produced from an F-1 hybrid between distantly related cultivars of Nipponbare (japonica type) and Milyang 23 (indica type). RFLP analysis revealed that ten and eleven of the 50 markers in the two AC-derived populations showed distorted segregation ratios from the theoretical ratio of 1:1. Parental alleles were not randomly transmitted from the F-1 plant to the AC-derived plants. Additionally, the segregation ratios of seven and six RFLP markers, respectively, were distorted both from the 1:1 ratios and from the observed ratios in the F-2 population. The chromosomal regions involving these markers were on chromosomes 1, 3, 7, 10, 11 and 12. Some of the regions were different in the two AC-derived populations. Non-random assortment of parental alleles might be influenced by anther culture methods.