Parthenogenetic reproduction has only previously been demonstrated in two species of agromyzid leafminer flies (Diptera: Agromyzidae), both from the genus Phytomyza. The plantain leafminer, Phytomyza plantaginis, is Palaearctic in origin, and bisexual populations have been observed in this region. However, historically, only females had been collected in the Australasian, Nearctic, Neotropical and Oceanian regions. Here, we show that southern Australian samples of this species from Plantago spp. can be composed of both parthenogenic and bisexual populations. In sites around Melbourne, males were present, although the sex ratio was female biased, with females comprising 75% of sampled individuals. In contrast, males were absent from collections in western and northern Victoria, Australian Capital Territory, New South Wales and Western Australia. Female P. plantaginis flies were reared from three Plantago host plants (Plantago lanceolata, Plantago major and Plantago coronopus), although females were particularly common from Pl. lanceolata. Insect cages set up with leafminers from sites where only females occurred produced only females, while cages with leafminers from sites where males occurred produced both sexes and mating was observed. Individuals from all populations were infected by Wolbachia endosymbionts, suggesting that the presence/absence of Wolbachia is not directly responsible for the parthenogenesis. However, there was an association between a mtDNA variant (PP.02) and sexual reproduction, in that this variant was absent in areas where only females were collected but present in all males and some females from areas with sexual populations. The mechanism responsible for parthenogenesis in P. plantaginis remains unknown but is linked to maternal factors.