Acrocerid flies are endoparasitoids of spiders that are distributed in all ecosystems except deserts and the poles. This group was historically classified into three subfamilies. However, molecular analyses have indicated two new subfamilies. One of these is Cyrtinae Newman, which is made up of two clades: the north, with Palearctic species, and the south, with Chilean species (except for the fossil genus Villalites Hennig). In the southern clade, endemic genera Holops Philippi and Villalus Cole have been poorly described over time by different authors. The aim of this study is to re-describe the known species of Holops and Villalus and describe three new Holops species based on collected material: Holops grezi sp. nov., Holops anarayae sp. nov., and Holops pullomen sp. nov. In addition, an identification species key with updated distributions for all species and the first record of Villalus inanis from Argentina is also provided. New species are mainly characterized by the length of flagellum; colors of the postocellar lobe and thoracic segments, pilosity and legs; wing cells and veins, and shape of the abdomen. Holops and Villalus do not share morphological traits with any other known extant genus of Acroceridae in the southern hemisphere. The real biodiversity of this group must be evaluated with a molecular phylogeny in the future. In addition, the new species in the Pehuén and Valdivian forest provinces, areas considered to have been sheltered during the last glacial period, promoting speciation in several taxa. New and cryptic Holops species open the doors to disentangling the real biodiversity of spider flies in Chile.
Keywords: Holops; Maule province; Pehuén and Valdivian districts; Spider flies; Valdivian forest province.