The activity of acid phosphatase in root tips is an important index for screening phosphorus efficiency of soybean varieties. Mining for candidate genes with acid phosphatase activity and exploiting their functional markers will bring great significance for identifying the phosphorus-efficient genes, unlocking the molecular mechanism of phosphorus utilization as well as breeding for new phosphorus-efficient soybean varieties. In this study, two DNA libraries with bulked pools of segregants from a F12 recombination inbred line population were constructed for next generation sequencing. 268 significant SNPs were obtained by SLAF-BSA technique. That included 12 gene-based non-synonymous mutations, seven of which were found between parents and five of which were found between offspring pools. Particularly, two candidate regions were identified, where 79 acid phosphatase-related genes were resided. Four genes are allocated from 20138271 to 20268154 on chromosome 3 and 75 genes are allocated from 1436648 to 15526449 on chromosome 17. Furthermore, a genetic marker GMsnp-B, which targets to the non-synonymous mutation gene Glyma.17G166200.1, was developed and tested using 169 soybean cultivars. The coincidence rate with phenotype activities in root tips was 82.8%.