This is the first report of Hoplotylus femina in Spain. It was collected from the rhizosphere and roots of common alder (Alnus glutinosa) in La Alberca, Salamanca Province, in Spain. The morphology and morphometrics of the Spanish population of H. femina agree closely with the original description of the species and other descriptions from The Netherlands, USA, New Zealand, Poland, Japan and Mexico. Only small differences in some morphological characters were found probably due to geographical intraspecific variability and/or different methods of fixing nematodes. This study provides new molecular markers for species identification (D2-D3 expansion regions of 28S and ITS-rRNA genes) and a new phylogenetic position for the D2-D3 region marker. Maximum likelihood analysis using the Shimodaira-Hasegawa test did not support the inclusion of Radopholus and Hoplotylus in the Pratylenchidae and both genera were more related phylogenetically to Hoplolaimidae.