Teat number is an important economic trait in the pig breeding industry and is directly related to the livability and growth status of piglets. By applying genetic technology, it may be possible to increase the number of teats and thus enhance production. Previous research has shown that teat number exhibits moderate levels of heritability that could be accelerated by the use of a genetic marker, such as microsatellites. Based on our preliminary sequencing data, the present study describes the use of time-of-flight mass spectrometry (TOF-MS) to identify the locations of four new microsatellites in Qinghai Bamei pig (n=256), an indigenous breed of Chinese pig. TOF-MS demonstrated that the L1-(GT)n locus had three alleles: 152 bp, 164 bp, and 166 bp. The three other loci, L2-(AC)n, L3-(TC)n, and L4-(TGA)n, all had two alleles. Alignment of our sequencing data with the Sus scrofa reference genome (Sscrofa11.1) revealed that the L1-(GT)n microsatellite, which features a GT repeat motif, was located in the retinoic acid induced 2 gene (RAI2) on chromosome X in Sus scrofa (SSCX). Analysis further revealed that the L2-(AC)n locus, featuring AC repeats, and the L3-(TC)n locus, featuring TC repeats, were located in intergenic regions on SSC14 and SSC5, respectively. TGA repeats within the L4-(TGA)n locus were located in the inner mitochondrial membrane peptidase subunit 2 gene (IMMPL2) on SSC18. Further association analysis revealed that the L1-(GT)n locus was significantly associated with teat number (p < 0.05) and that the 152-bp allele exhibited the most positive association. Our data indicate that the L1-(GT)n microsatellite could represent a potential DNA marker with which to increase the number of teats. Thus could help to improve production in the pig breeding industry.