By using conventional bacteria medium (BPM), inorganic phosphate-solubilizing bacteria (PSB) medium (IPM) and organic PSB medium (OPM), culturable microbial colonies were collected after third serial passage on agar plates. High-throughput sequencing analysis of phoD gene as molecular target were employed to evaluate the diversity differences of PSB in the background soil and colony enrichments from different media. The results showed that phoD gene sequences obtained from high-throughput sequencing could largely cover phoD-related PSB communities in the background soil and colony enrichments, and the Goods coverage index exceeded 98.3%. α diversity of phoD gene in colony enrichments were significantly lower than the background soil, and Shannon and Simpson indices of phoD gene were the lowest in IPM, but had no significant difference between BPM and OPM. Chao 1 index of phoD gene showed no significant difference in different media, accounting for 5.3%–8.4% of the background soil. According to the taxonomic annotation of phoD gene, 34 genera of PSB were detected in the background soil and the proportion of culturable PSB were only about 26.5%–41.2%. Among the background soil and the colony enrichments from different media, there were 6 common genera of PSB, which usually dominated in the culturable PSB communities, including Pseudomonas, Bradyrhizobium, Cupriavidus, Sinorhizobium, Xanthomonas and Actinoplanes,. Further LEfSe (LDA effect size) analysis showed that 4 genera above had significant differences among different media, Cupriavidus was significantly enriched in both IPM and OPM, while Pseudomonas, Bradyrhizobium and Xanthomonas were significantly enriched in both BPM and OPM. Moreover, Burkholderia was only detected in OPM. Finally, at the genus level, lots of phoD gene sequences couldn’t be clearly classified at present which accounted for 0.13% to 59.6% of the total phoD sequences. In conclusion, by combining traditional microbial culture technology with high-throughput sequencing technology, this study quantitatively analyzed the influence of medium components on phoD gene diversity of soil culturable PSB, and meanwhile revealed the culturable and physiological characteristics of phoD-related PSB, which can provide a reference for the exploration and utilization of soil PSB resources. In the future, the innovation of culture technology is urgently needed to isolate and identify more PSB for enhancing the overall understanding of PSB resources in the environment.