The paper presents the results of genotypic differentiation and antimicrobial susceptibility of Y. enterocolitica O:9 isolates that originated from cows and pigs positive in serological reactions for brucellosis, and also from the animals, which were serologically negative. The genetic relationship between Y. enterocolitica O:9 isolates originating from different sources was determined by the use of ERIC-PCR, and resulted in detection of 6 to 13 DNA amplicons of different size. The clonal analysis was based on dendrogram created by Unweighted Pair Group Method with arithmetic mean and Jaccard’s coefficient, which enabled to divide Y. enterocolitica O:9 isolates into 16 different clonal groups. Among all Y. enterocolitica O:9, MIC value was >32 mg/L for the ampicillin, ≤0.008 mg/L for ciprofloxacin, ≤8 mg/L for sulphametoxazole, ≤2 mg/L for colistin, and ≤1 mg/L for tetracycline. The wide range of MIC for ceftazidime (≤0.25-2 mg/L) and cefotaxime (≤0.06-1 mg/L) among Y. enterocolitica O:9 isolates was also observed. No significant differences were observed between MIC values of Y. enterocolitica O:9 isolates originating from animals serologically positive for brucellosis, and the isolates from cows and pigs, which provided serologically negative reactions.