The genus Tulostoma Pers. (Order Agaricales; Basidiomycota) is characterized by a globose spore-sac, where the spores are formed, attached onto a stipe; they are known commonly as stalked puffballs. The sporesac is formed by the peridium that envelopes the gleba; when spores are mature, they are released via an opening found on the apical part of the sac called a mouth or stoma that can be tubular, circular, or elliptical; fibrillose or fimbriate; or a simple crevice. The current genus was initially divided into two sections: Eutylostoma and Schizostoma (Fischer, 1900, 1933; Petri, 1909; Fries, 1921), which are differentiated by mouth morphology. Pouzar (1958) offered a more precise classification based on the morphology and mode of opening of the exoperidium, as well as the morphology of the mouth and stipe, proposing four sections: Brumalia, with tubular mouth; Poculata, with mouth fibrillose; Fimbriata, with fimbriate mouth; and Volvulata, with irregular mouth. Wright (1987) supplemented Pouzar's classification with primary and secondary micromorphological characteristics. The primary characteristics are the endoperidium form, size, and color; exoperidium color, persistence, and decay; mouth form; and spore size and ornamentation. Secondary characteristics are stipe size, and color and morphology of stipe surface; thickness of capillitium hyphae; and morphology of capillitium transverse septa. In Wright's classification two subgenera are included: Tulostoma Pers. and Lacerostoma J. E. Wright, encompassing 137 species worldwide. As pointed out by Cunningham (1925), Moreno et al. (1992) considered that this genus consists of a taxonomic complex, since some characters are not always easy to differentiate. The genus has been studied in various areas of Europe, Asia Minor, and Africa. In the work of Kreisel (2001), 28 species were cited in Europe, 14 in Asia Minor, and 38 in Africa. In the Mediterranean area, Calonge et al. (2007) described 22 species for Spain, while Tkalčec et al. (2005) and Sesli and Denchev (2008) reported only five species in Croatia and Turkey. In Macedonia, based on morphological identification, Karadelev and Rusevska (2009) confirmed four species: Tulostoma brumale Pers., T. fimbriatum Fr., T. melanocyclum Bres., and T. squamosum (J.F. Gmel) Pers. (Table). Wright (1987) reported, for the first and only time, T. caespitosum Trab. Molecular analysis based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA), accepted as the DNA barcode for fungi (Schoch et al., 2012), provides valuable information for species identification. Recently, Jeppson et al. (2017) demonstrated that the ITS region is very useful to discriminate Tulostoma species in Europe; in this paper the barcode sequence was obtained Abstract: With the aim of clarifying the number of Tulostoma species in Macedonia, and to verify some previous collections, molecular analyses were carried out on four morphologically identified species collected in different habitats (at the edges of oak or juniper forests, pine ...