The Amphibia are considered the most threatened vertebrate class globally, yet in Brazil they are also one of the more diverse and species rich groups. Although, in recent years there has been strong focus on amphibian related research, their parasites have not received the same attention. In Brazil, only a single species of
Hepatozoon
, namely
H. leptodactyli
(Lesage, 1908) Pessoa, 1970, has been described from anuran hosts. The present study aimed to describe three new species of
Hepatozoon
parasitising
Leptodactylus labyrinthicus
and
Leptodactylus latrans
from Mato Grosso State, Brazil. From 66 anurans screened for haemogregarines, four belonging to the Leptodactylidae were found positive for species of
Hepatozoon
. Based on the morphological analysis of peripheral blood gamonts and spleen and liver tissue meronts, three different morphotypes of
Hepatozoon
spp. were identified. Morphotype 1 (M1) and morphotype 2 (M2) in
L. labyrinthicus
and morphotype 3 (M3) in
L. latrans.
Molecular data based on partial 18S rDNA sequences revealed an interspecific divergence, between the species ranging from 0.43% to 1.16%. Phylogenetic analysis recovered isolates from the present study monophyletic with other isolates from Brazilian reptile and anuran hosts, sister to a clade comprising species isolated from African, North American and European reptile and anuran host species. Thus, using morphological and molecular analysis three new species infecting Brazilian Leptodactylidae anurans were identified and described. This study increases the knowledge of Brazilian anurans blood parasites and demonstrates the importance of using integrative approaches for diagnosis of hemoparasites.