The genus Longidorus currently comprises 176 species of polyphagous plant ectoparasites, including eight species that vector nepoviruses. Longidorus is one of the most difficult genera to accurately identify species because of the similar morphology and overlapping measurements and ratios among species. Sequences of ribosomal RNA (rRNA)-genes are a powerful level-species diagnostic tool for the genus Longidorus. From 2015 to 2019, a nematode survey was conducted in vineyards and agro-forest environments in Portugal. The populations of Longidorus spp. were characterized through an integrative approach based on morphological data and molecular phylogenetic analysis from rRNA genes (D2-D3 expansion segments of the 28S, ITS1, and partial 18S), including the topotype of L. vinearum. Longidorus bordonensis sp. nov., a didelphic species recovered from the rhizosphere of grasses, is described and illustrated. Longidorus vineacola, with cork oak and wild olive as hosts, is also characterized. This is the first time that L. wicuolea, from cork oak, is reported for Portugal. Bayesian inference (BI) phylogenetic trees for these three molecular markers established phylogenetic relationships among the new species with other Longidorus spp. Phylogenetic trees indicated that i) L. bordonensis sp. nov. is clustered together with other Longidorus spp. and forms a sister clade with L. pini and L. carpetanensis, sharing a short body and odontostyle length, and elongate to conical female tail, and ii) all the other species described and illustrated are phylogenetically associated, including the topotype isolate of L. vinearum.