The genetic diversity analysis of 135 rice germplasm resources collected from different altitudes in Yunnan province, P.R. China, were genotyped by using the gene chip GSR40K. The rice germplasm resources at different altitudes were found with rich genetic diversity, and they were divided into five subgroups: indica, partial indica, intermediate, japonica and partial japonica. Eighty-two genes with expected breeding value were genotyped by haplotype markers and functional markers. All accessions were found with the genes related to the grain shattering. Nearly 70% of rice varieties were detected with rice blast resistant genes, whereas only a small proportion of accessions containing insect resistance and fragrance genes were identified. The accessions in this collection were divided into seven subgroups by cluster analysis and principal component analysis. The differentiation of each marker site among the 7 subgroups was evaluated by the Gst value of genetic differentiation index, indicating that there was high genetic differentiation among the 7 subgroups. Moreover, at least 10% of the genomic segments in this collection are completely different, and only 7.5% are frequently communicated and fixed, suggesting an extremely low frequency of gene exchange between different subpopulations. Based on the difference of altitude among subpopulations, the differential genomic regions among populations were proposed associating with altitude adaptability. Collectively, these results provided scientific basis for effective protection and efficient utilization of rice resources in rice breeding.