Pulsed-field gel electrophoresis (PFGE) represents the gold standard among band-based methods for the molecular typing of Bartonella henselae. SmaI and NotI have been frequently used for typing B. henselae by PFGE. However, their appropriateness for the analysis of genetic relatedness among B. henselae isolates has not been assessed systematically hitherto. Aim of the present study was to evaluate SmaI, NotI, and three additional endonucleases for typing B. henselae isolates by PFGE and to compare the PFGE results with multi-locus sequence typing (MLST) data. Twenty B. henselae isolates from different sources and geographic regions were analysed. PFGE analysis upon restriction with SmaI, ApaI, Eco52I, and XmaJI revealed six, five, four, and five different PFGE types, respectively, whereas restriction with NotI revealed 13 PFGE types. Five sequence types (STs) were obtained by MLST. The overall concordance between PFGE types obtained with SmaI, ApaI, Eco52I, XmaJI and STs was high. In contrast, NotI-derived types did not correlate with other PFGE types or STs, indicating that NotI is not an appropriate enzyme for PFGE typing of B. henselae. By combining PFGE results obtained with SmaI, ApaI, Eco52I, XmaJI with STs, the isolates could be assigned to five distinct clonal lineages, including the clones Houston-1, Marseille, CAL-1, and Berlin-2. These data indicate that PFGE and MLST are discriminatory and reliable for molecular typing of B. henselae isolates to the strain level. Combination of PFGE and MLST may be useful for further epidemiological studies on B. henselae.