Oryza glumaepatula and Oryza barthii are AA-genome wild species in Oryza genus. In order to effectively mine and transfer beneficial genes from these wild species in genome-wide by distant hybridization, in the present study, the chromosome single-segment substitution lines (SSSLs) were developed using O. glumaepatula and O. barthii as donors and elite indica cultivar, HJX74, as a recipient. A total of 168 homozygous SSSLs of O. glumaepatula (GLU-SSSLs) and 95 homozygous SSSLs of O. barthii (BAR-SSSLs) were developed. In 168 GLU-SSSLs and 95 BAR-SSSLs, the total lengths of the substituted segments distributing on 12 chromosomes were 3636.35cM and 1731.65cM, respectively. Accordingly, the mean length of each substituted segment was 21.64cM in GLU-SSSLs and 18.23cM in BAR-SSSLs. The total coverage lengths of the substituted segments on rice genome were 80.15% in GLU-SSSLs and 54.67% in BAR-SSSLs. To evaluate the utilization potential of these SSSLs, four yield-related traits, heading date, plant height, seed-setting rate, and 1000-grain weight, were investigated in the SSSLs over two seasons. Significant variations of the traits were observed among the SSSLs. At P0.05 level, a sum of 28 stable QTLs in GLU-SSSLs and 53 in BAR-SSSLs were identified. Among them, the additive effects of 12 QTLs contributed averagely more than 10% for phenotypic variations, viz., qPHg1-2, qGWTg9-2, qHDb1-1, qPHb1-2, qPHb1-3, qPHb7-1, qPHb7-2, qPHb8-1, qPHb10-1, qPHb12-2, qSSb7-1, and qSSb10-2. Nine pairs of QTLs in the two donors were comparatively mapped on chromosomes 1, 5, and 10, which means the same QTLs or alleles existing in O. glumaepatula and O. barthii have been detected and transferred to SSSLs. Besides, qGWTg1-1, qGWTg4-1, and qGWTb1-1 have shown positive additive effects, implying alleles from the two wild donors of these QTLs have the potential in increasing 1000-grain weight in SSSLs. The SSSLs library of the wild species is a valuable resource for rice germplasm innovation and breeding.