Nymphal and larval Amblyomma cordiferum ticks, a relatively rare species, were collected from the Malaysian house rat (Rattus rattus diardii) in Peninsular Malaysia. Redescription and molecular analysis of nymphs and larvae, based on the 12S rRNA, 16S rRNA, and COI genes, revealed divergence from A. cordiferum in Taiwan, suggesting that the Taiwanese tick specimens may represent a different taxon. Molecular analysis of the pathogens in these specimens revealed three sequences of Rickettsia closely related or identical to Rickettsia raoultii (99.71-100%), two sequences of relapsing fever Borrelia identical to Borrelia theileri, and four sequences of Bartonella identical to Bartonella phoceensis. This study also identifies a new host record for A. cordiferum in R. r. diardii and reports the first detection of Rickettsia, Borrelia, and Bartonella in this tick species.