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Background  Understanding and identifying the factors responsible for genetic differentiation is of fundamental importance for efficient utilization and conservation of traditional rice landraces. In this study, we examined the spatial genetic differentiation of 594 individuals sampled from 28 locations in Yunnan Province, China, covering a wide geographic distribution and diverse growing conditions. All 594 accessions were studied using ten unlinked target genes and 48 microsatellite loci, and the representative 108 accessions from the whole collection were sampled for resequencing.    Results  The genetic diversity of rice landraces was quite different geographically and exhibited a geographical decline from south to north in Yunnan, China. Population structure revealed that the rice landraces could be clearly differentiated into  japonica  and  indica  groups, respectively. In each group, the rice accessions could be further differentiated corresponded to their geographic locations, including three subgroups from northern, southern and middle locations. We found more obvious internal geographic structure in the  japonica  group than in the  indica  group. In the  japonica  group, we found that genetic and phenotypic differentiation were strongly related to geographical distance, suggesting a pattern of isolation by distance (IBD); this relationship remained highly significant when we controlled for environmental effects, where the likelihood of gene flow is inversely proportional to the distance between locations. Moreover, the gene flow also followed patterns of isolation by environment (IBE) whereby gene flow rates are higher in similar environments. We detected 314 and 216 regions had been differentially selected between Jap-N and Jap-S, Ind-N and Ind-S, respectively, and thus referred to as selection signatures for different geographic subgroups. We also observed a number of significant and interesting associations between loci and environmental factors, which implies adaptation to local environment.    Conclusions  Our findings highlight the influence of geographical isolation and environmental heterogeneity on the pattern of the gene flow, and demonstrate that both geographical isolation and environment drives adaptive divergence play dominant roles in the genetic differentiation of the rice landraces in Yunnan, China as a result of limited dispersal.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12284-021-00497-6.

Genetic differentiation and restricted gene flow in rice landraces from Yunnan, China: effects of isolation-by-distance and isolation-by-environment