A multilocus sequence typing (MLST) scheme was developed to evaluate the genetic diversity and background of Staphylococcus saprophyticus strains of food and clinical origins. A total of 48 isolates from human urine samples (21 isolates), Doenjang (9 isolates), Myeolchi-jeotgal (6 isolates), Saeu-jeotgal (3 isolates), and sausages (9 isolates) were subjected to MLST using internal fragments of 7 housekeeping genes: aroE, dnaJ, glpF, gmk, hsp60, mutS, and pta. MLST analysis resulted in 26 sequence types (STs), and the eBURST algorithm clustered the STs into 4 clonal groups (CGs) and 6 singletons. The predominant STs, ST11 (16.7 %, 8/48), and ST7 (10.4 %, 5/48), belonged to the major CG, CG1 (12 STs, 60.4 %, 29/48), and comprised isolates from Doenjang and urine. CG2 and CG3 comprised isolates from sausages, while CG4 included isolates from Myeolchi-jeotgal and Saeu-jeotgal. Twenty of the S. saprophyticus isolates exhibited resistance to erythromycin, lincomycin, or tetracycline. Lincomycin resistance was identified from the ST10–12 isolates, all of which are variants of ST08 and were isolated from Doenjang and human urine samples. This MLST scheme established the genetic diversity within S. saprophyticus, and clustering of the STs using eBURST revealed a correlation between the genetic backgrounds and the origins of isolates, and a link between genetic background and acquisition of lincomycin resistance.