A large collection of Staphylococcus aureus including a. 745 clinically significant isolates that were consecutively recovered from human infections during 2012-2013, b. 19 methicillin-susceptible (MSSA), randomly selected between 2006-2011 from our Staphylococcal Collection, c. 16 human colonizing isolates, and d. 10 strains from colonized animals was investigated for the presence and the molecular characteristics of CC398. The study was conducted in Thessaly, a rural region in Greece. The differentiation of livestock-associated clade from the human clade was based on canSNPs combined with the presence of the φ3 bacteriophage and the tetM, scn, sak, and chp genes. Among the 745 isolates, two MRSA (0.8% of total MRSA) and thirteen MSSA (2.65% of total MSSA) were found to belong to CC398, while, between MSSA of our Staphylococcal Collection, one CC398, isolated in 2010, was detected. One human individual, without prior contact with animals, was found to be colonized by a MSSA CC398. No CC398 was identified among the 10 S. aureus isolated from animals. Based on the molecular markers, the 17 CC398 strains were equally placed in the livestock-associated and in the human clades. This is the first report for the dissemination of S. aureus CC398 among humans in Greece.