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In 2020, adult hard ticks (males and females) were collected from great horned owls [Bubo virginianus(Gmelin, 1788)] in the coastal region in southern Brazil. The engorged females were allowed to oviposit in the laboratory and hatched larvae could be obtained. Analyses of the external morphology of the adult ticks revealed that they represent a new species, which was named Amblyomma monteiroaen. sp. Partial sequences of the mitochondrial 16S rRNA gene and the nuclear second internal transcribed spacer (ITS2) were generated from a male and a female. Their 16S rRNA haplotypes were identical to each other and closest (96% identity) to corresponding sequences of Amblyomma parvitarsumNeumann, 1901, and 90% identical to Amblyomma neumanniRibaga, 1902. Their ITS2 haplotypes were 95.8 to 96.0 identical to the single ITS-2 partial sequence of A. parvitarsumavailable in GenBank. In the phylogenetic trees inferred by both 16S rRNA and ITS2 partial sequences, A. monteiroaen. sp. formed a clade with A. parvitarsum,with A. neumannibranching sister to this clade. Amblyomma monteiroaen. sp. is genetically and morphologically related to A. parvitarsum.Both tick species are unique in combining the following morphological characters: scutum extensively ornate; eyes rounded and bulging; coxa I with two moderate pointed spurs, the external longer than the internal; a single triangular short spur on coxae II-III; presence of two spines on the tibia of legs II-IV; hypostomal dentition 3/3, trochanters without spurs. However, the males of the two species can be separated by specific features in palps and festoons, whereas the females differ in specific features of the coxal spurs. The larva of A. monteiroaen. sp. can be morphologically distinguished from A. parvitarsumonly by morphometry, with the former species being slightly smaller. Currently, A. monteiroaen. sp. is restricted to southern Brazil, and the only known host is B. virginianus(Strigiformes: Strigidae). The present study increases the AmblyommaBrazilian fauna to 34 species.