Sample collection and marker selection are of primary importance in population genetic studies, and can greatly influence the analysis and interpretation of the data obtained. Microsatellite DNA sequences are the most revealing DNA markers available so far for inferring population structure and have been widely employed in genetic studies of populations．Nevertheless，few studies have specifically examined the effects of sample size and loci number on various genetic diversity measures in population genetic work using these markers．Here，we examined this issue using experimental data from an analysis of genetic diversity of wild populations of grass carp at 45 polymorphic microsatellite loci. The following genetic diversity measures were studied: number of alleles per locus (Na), effective number of allele per loci (Ne)，observed hererozygosity (Ho) and expected heterozygosity (He)．We also tried to infer the theoretical minimum sample size and loci number needed for population genetic studies with microsatellite loci. The results are as follows: (1) Na and Mean number of allele over loci (MNA) are significantly affected by sample size. It increase according to the raise of the sample size, only when the sample size exceeds 40 it tends to be stable. The significant correlation between sample size and Na was observed and the correlation curve is Y=3.8403+0.6376, r2=0.967．However, there is no significant correlation between the mean value of Ne, Ho, He and the sample size. Mean value of Ho, He fluctuate with the raise of the sample size, only when the sample size exceeds 40 it manifest a stable trend. (2) Evaluating of different genetic parameters with the makers of different polymorphism information content, a significantly different result were acquired. When PIC>0.5, Na, Ne, Ho, He were 3.6, 1.5934, 0.2824, 0.3540, While PIC<0.5, Na, Ne, Ho, He were 9.5, 5.9209, 0.8497, 0.8242 respectively. Accordingly, marker selecting strategy has much greater impact on evaluation of genetic diversity. (3) According to the rise of loci number, the value of genetic parameter could be divided into three stages: ① Descending stage: loci number between 0−5, values of Na, Ne, Ho, He indicated descending trend. ② Loci number between 5−10, values of Na, Ne, Ho, He demonstrate ascending trend. ③ When loci number exceeded 15 values of Na, Ne, He tend to be stable, while only when loci number exceeded 25 the value of Ho tend to be stable. Analysis above proved that not only the sample size and loci number but also the polymorphism information of microsatellite markers have significant effect on the estimation of population genetic diversity. So, we suggested that when microsatellite markers were used to estimate genetic diversity of population the minimum loci number and sample size are needed to exceed 25 and 40, respectively. Moreover, the conclusion above has been used to evaluate the genetic diversity of wild population of grass carp, values of the parameter were Na=7.26、Ne =4.21、Ho=0.73、He=0.68, which demonstrated that has a relatively high diversity, a totally different conclusion from the former studies. Therefore, further researches are necessary for the evaluation of genetic diversity of geographically different population of grass carp.