Mycoplasma gallisepticum (MG) is the most pathogenic avian mycoplasma species. It affects commercial and noncommercial poultry and wild birds. Current MG sequence typing methods rely on the partial sequence of one or more surface antigen genes. Multilocus sequence typing (MLST), a widely used typing method for many human and animal pathogens, relies on conserved housekeeping genes. Recently, MLST assays have been developed for Mycoplasma synoviae (MS) and Mycoplasma iowae. Additionally, a whole genome–based core genome MLST (cgMLST) assay has been developed for MG and MS. However, cgMLST can be implemented only on pure isolates and cannot be applied to clinical samples. Here, we have developed a seven-locus–based MLST scheme for MG that can be applied directly on clinical samples without the need for isolation. The seven loci were selected from 425 genes recently used for the cgMLST assay. A total of 101 diverse MG samples, including isolates and clinical samples, were typed with the newly developed seven-locus MLST. The phylogeny and discriminatory power of the seven-locus MLST were evaluated and compared with the cgMLST and gene-targeted sequencing methods currently used for MG sequence typing. The seven-locus MLST provided optimum discriminatory power and congruent phylogeny to cgMLST. Additionally, a database for MG MLST was created and is currently available for public use online. This assay will increase accessibility to MG sequence typing and provide a stable and expandable nomenclature compatible with cgMLST. The seven-locus MLST assay represents an important tool for epidemiologic investigation of MG that can contribute to better control and eradication efforts.