ABSTRACT The present study evaluated the molecular detection and identification of Rickettsia species in 83 ticks collected in Sardinia, Italy. Fifteen ticks were PCR-positive using gltA-specific and ompA-specific primers, leading to the identification of Rickettsia aeschlimannii in Hyalomma marginatum marginatum, R. massiliae in Rhipicephalus turanicus and in Rhipicephalus sanguineus, and a new rickettsia, previously referred to as PoTiRb169 in Portugal, in four Rhipicephalus turanicus. This new species was further characterized by amplification and sequencing of three additional genes (ompB, sca4 and rrs). Using the current criteria to name a rickettsia, this uncultivated rickettsia can be given a Candidatus status, and we propose to call it 'Candidatus Rickettsia barbariae'. The detection of three tick-borne rickettsiae in Sardinia raises the possibility that many cases of spotted fever considered by clinicians and health authorities as Mediterranean spotted fever due to R. conorii could, in fact, be due to other rickettsiae, including those found in this study. Analysing skin biopsies of inoculation eschars in patients with spotted fever would be, together with continuing entomological surveys, the best way to increase our knowledge of tick-borne rickettsioses in Sardinia and more generally in the Mediterranean basin.