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<i>Bartonella</i> are vector-borne parasitic bacteria that cause zoonotic infections in humans. One of the most common infections is cat-scratch disease caused by <i>Bartonella henselae</i> and <i>Bartonella clarridgeiae</i>. Cats are the major reservoir for these two species of bacteria, while cat fleas are vectors for the transmission of infection agents among cats. The aim of the present study was to investigate the presence of <i>Bartonella</i> infections in stray and pet cats and in cat fleas in Lithuania. Blood samples were taken from 163 cats presented in pet clinics and animal shelters. A total of 102 fleas representing two species, <i>Ctenocephalides felis</i> and <i>Ctenocephalides canis</i>, were collected from 12 owned cats that live both outdoors and indoors. <i>Bartonella</i> DNA in samples was detected using a nested PCR targeting the 16S–23S rRNA intergenic spacer (ITS) region. <i>Bartonella</i> DNA was detected in 4.9% (8/163) of the cats and 29.4% (30/102) of the fleas. Sequence analysis of the ITS region showed that the cats and fleas were infected with <i>B. henselae</i>, <i>B. clarridgeiae</i> and <i>Bartonella</i> sp., closely related to <i>B. schoenbuchensis</i>. This study is the first report on the prevalence and molecular characterization of <i>Bartonella</i> spp. in cats and cat fleas in Lithuania.