Crop germplasm resources are the material basis of cultivar improvement. Along with the development of molecular biology technology, rice breeding has developed to a senior stage, which combines molecular marker assisted selection with agronomic traits selection. It is necessary that evaluation of germplasm resources met the needs of molecular assistant breeding and assisted by molecular method. 24 SSR markers recommended by China National Rice Research Institute was adopted to analyze the polymorphisms of 45 rice germplasm resources(32 restorer lines and 12 maintainer lines). Enhancement of germplasm was realized according to the differences in genetic similarity coefficient and agronomic traits. Molecular marker PCR-AccI which could detect the content of amylose in middle generation of the breeding was utilized for assisted selection; and germplasm lines with high quality was created. The results showed the genetic similarity coefficient of the 32 restorer lines and 12 maintainer lines was 0.5244~0.8415(averaged at 0.6965) and 0.4783~1(averaged at 0.6542), respectively. Using R3076, Zhonghui 8006, 229 and Jufeng A/B as parent, new restorer line R7606 and cytoplasmic male sterile (CMS) line 229A were obtained. Based on the detection results of rice quality, 11 varieties of R7606 line and 1 of 229A line achieved grade Ⅱ of national rice quality standard(GB/T 17891-1999); and 3 varieties of 229A achieved grade Ⅰ of national rice quality standard. The result of field observation and hybrid testing experiments revealed that R7606 and 229A lines had elite agronomic traits and good general combining abilities, and could be utilized as parents in hybrid-rice breeding. It had also been discussed how to evaluate and apply excellent resources in rice genetic improvement and germplasm enhancement to expand the genetic background and diversity of germplasm resources and promote sustainable development of rice breeding and production.