Perilla seed oil has been attracting attention in South Korea as a health food. Five fatty acids of 100 Perilla accessions were identified as follows: palmitic acid (PA) (5.10–9.13%), stearic acid (SA) (1.70–3.99%), oleic acid (OA) (11.1–21.9%), linoleic acid (LA) (10.2–23.4%), and linolenic acid (LNA) (54.3–75.4%). Additionally, the 100 Perilla accessions were divided into two groups (high or low) based on the total fatty acid content (TFAC). By using an association analysis of 40 simple sequence repeat (SSR) markers and the six Perilla seed oil traits in the 100 Perilla accessions, we detected four SSR markers associated with TFAC, five SSR markers associated with LNA, one SSR marker associated with LA, two SSR markers each associated with OA and PA, and four SSR markers associated with SA. Among these SSR markers, four SSR markers (KNUPF14, KNUPF62, KNUPF72, KNUPF85) were all associated with TFAC and LNA. Moreover, two SSR markers (KNUPF62, KNUPF85) were both associated with TFAC, LNA, and OA. Therefore, these SSR markers are considered to be useful molecular markers for selecting useful accessions related to fatty acid contents in Perilla germplasm and for improving the seed oil quality of Perilla crop through marker-assisted selection (MAS) breeding programs.