Abstract Knowledge about molecular epidemiology of B. henselae is important for recognizing the geographical distribution of strains and identification of isolates virulent for humans. Eleven Polish feline B. henselae isolates were typed, using 2 different techniques: pulse-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA). PFGE analysis distinguished 6 different PFGE types, with subtypes within 3 of them, whereas 10 MLVA types were assigned. Global diversity index (D.I.) for MLVA equaled 0.93. For 7 isolates, the results of MLVA confirmed cluster assignments based on PFGE. Both PFGE and MLVA results were in accordance with epidemiological data. Although PFGE has been previously demonstrated to be a suitable method for the differentiation of B. henselae isolates/strains, our results show the superiority of MLVA over PFGE with respect to higher discriminatory power, distinguishing genotypes I and II isolates, easier analysis of results, and possibility to compare the numerical data obtained by different laboratories. With MLVA, 7 new profiles were observed, compared to previous results from around the world; whereas 3 known profiles were previously described mainly in European B. henselae isolates. Our results confirm that some VNTR profiles can be used as specific geographical markers.