Analysis of genetic diversity in maize germplasm is of great importance to conserving and utilizing these valuable genetic resources. However, when a large-scale analysis of genetic diversity in maize landraces and breeding populations which are genetically heterogeneous is conducted, the choice of sampling methods will seriously affect research results and efficiency. To solve the problem of sampling methods, individuals and bulking samples of different individuals of two breeding populations and three landraces were selected to be molecularly characterized using microsatellite (SSR) markers. The results showed that there were differences of intra-population genetic variability among different populations. Different bulking samples with the same number of individuals showed similar results, but bulking samples with different individuals presented some different results to some extent and the differences seemed to associate with the extent of genetic variability of the entries. Considering both scientific significance and research feasibility, we suggest that one bulk from randomly chosen 30 individuals (or two bulks each from randomly chosen 15 individuals) representing one population be genotyped when genetic diversity of maize landraces and breeding populations is assessed based on molecular markers, e.g. SSR.