Red paddy soil is one of the most important typical soils in subtropical regions of China. Appropriate fertilizer application is an important management practice to improve soil fertility and quality. Here, four paddy soil samples under long-term fertilization practices, the first was control without any treatment (CK), the second treated with only urea (N), the third treated with urea and recycled with all the rice straw (NC) and the last treated with not only urea, but also calcium magnesium phosphate and potassium chloride (NPK), were collected. Total C and N, available N, P and K and soil pH were measured. 28, 31, 27, 22 unique profiles screened by RFLP were identified from nifH-CK, nifH-N, nifH-NC, nifH-NPK libraries, respectively. The results showed that there was highest microbial population genetic diversity of N sample while NC sample and NPK sample had little change comparing with CK sample.