Two broadly disjunct populations of Mishana Tyrannulet are diagnosably distinct, especially based on voice (Whitney et al. 2013e, with reference to audio recordings by Dan Lane). Available recordings document that the common whistled call of the San Martín population begins with a stronger downward component, and may be higher and thinner on average, than the population in the Nanay and Tigre basins in Loreto. Habitat preferences also may differ between the two populations, though it is unclear to what extent observed differences in habitat use simply reflect different habitat availability in the two regions, rather than a difference in preferences. Currently, however, the San Martín population lacks a name, and so Zimmerius villarejoi is monotypic.
On the basis of morphology and vocalizations, Mishana Tyrannulet generally is considered to belong to a group of related Zimmerius tyrannulets that also includes Chico's Tyrannulet (Zimmerius chicomendesi) and Red-billed Tyrannulet (Zimmerius cinereicapilla). The red bills of all three species are distinctive within the genus, and the species additionally share simple vocalizations consisting of short, sweeping whistles. A phylogenetic analysis of Zimmerius tyrannulets based on two mitochondrial loci recovered a strongly supported sister relationship between Red-billed Tyrannulet and the San Martín population of Mishana Tyrannulet; the analysis did not include Chico's Tyrannulet or the Loreto population of Mishana Tyrannulet (Rheindt et al. 2013). Thus, we currently lack a comprehensive assessment of relationships among the "red-billed" Zimmerius, but morphological and vocal data strongly suggest that Chico's Tyrannulet and both populations of Mishana Tyrannulet are closely related, with Red-billed Tyrannulet somewhat more distant. The phylogeny recovered by Rheindt et al. (2013) further suggests that the "red-billed" Zimmerius clade is basal to the remainder of the genus.