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Currently four species of Holochilus (Cricetidae: Sigmodontinae) are recognized. According to the literature, three species are recorded for Brazil: H. sciureus, H. brasiliensis and H. chacarius. Samples from western Brazil (Mato Grosso and Mato Grosso do Sul states) are usually reported as H. sciureus and, when referring to specimens from the Pantanal, as H. chacarius. However, the taxonomic status of specimens from this region has not been properly evaluated through detailed morphological analyses or voucher specimens. About 110 specimens of Holochilus deposited in Brazilian collections were qualitatively and quantitatively analyzed (univariate and multivariate tests). Our results suggested the existence of three species (H. sciureus, H. brasiliensis and H. chacarius) in Brazil, which present significant morphological and morphometric differences, thus confirming that the Mato Grosso and Mato Grosso do Sul samples, formerly misidentified as H. sciureus, belong to H. chacarius. This species differs from H. sciureus and H. brasiliensis by a series of pelage and skull characters, such as: the coloration of pelage, light orange-brown in the dorsum, light orangish in the flanks and white in the venter; ridges of the masseteric crest confluent at the level (or slightly above) of the mental foramen and at the anterior part of m1, alternated main molar cusps, lophids compressed and with acute outer margins (rarely prismatic), anteromedian fossetid labially displaced and subcircular (small to medium), metaflexid less developed (frequently not reaching the midline of the tooth), proto and hipoconid subrectangular in outline and transversely orientated, mesoflexid transversely orientated and mesoloph absent. The updated geographic distribution of Holochilus in Brazil shows that H. sciureus is present in the northern portion of the country, being the only species present at the Amazon and at the Caatinga and with limit in the Cerrado biome at the center of Goiás state. Holochilus brasiliensis is restricted to eastern Brazil, with its northern record at the Atlantic Forest of south Bahia. Holochilus chacarius occurs in the Pantanal areas of the Mato Grosso and Mato Grosso do Sul states. A new record extends the distribution of H. chacarius to the Cerrado biome at eastern Mato Grosso, confirming its presence in this biome

On the occurrence of Holochilus chacarius (Cricetidae: Sigmodontinae) in Brazil, with taxonomic notes on Holochilus species