The genus Matuxia Carbayo et al., 2013 currently comprises two species with distribution restricted to southeastern Brazil. In the present study, based on an integrative approach, we examine the genetic diversity within the genus and describe a new species, Matuxia tymbyra Rossi and Leal-Zanchet, sp. nov., representing a southern lineage of the genus. We employed one mitochondrial (cytochrome c oxidase subunit I, COI) and other nuclear (elongation factor 1a, EF-1a) markers to investigate the phylogenetic relationships within the genus. Maximum-parsimony analysis in a segment of the COI gene of 676 nucleotides showed 79 (11.68%) nucleotide positions exhibiting autapomorphic characters that support the occurrence of three independent evolutionary molecular operational taxonomic units in the genus. Similar evaluation for the dataset of the EF-1a gene showed a much smaller number of autapomorphies. Bayesian inference, maximum-likelihood and delimitation approaches, based on evolutionary models, showed that M. tymbyra is the sister species of the type-species of the genus, Matuxia tuxaua. They should be considered sibling species, only distinguishable based on details regarding eye arrangement and prostatic vesicle. The existence of 15 and 23 molecular autapomorphies, as revealed by maximum-parsimony analysis in a segment of the COI gene for M. tymbyra and M. tuxaua, respectively, allowed us to propose a molecular diagnosis for the new species, which is essential in cases of sibling species. The new species seems to be endemic from areas of Araucaria Forest in southern Brazil; the record augments the known distribution of the genus to the south.