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Recently, it has been shown that the stem nematode, Ditylenchus dipsaci (Nematoda: Anguinidae), is genetically more related to the gall-forming nematodes from genera Anguina, Heteroanguina, and Mesoanguina than to other members of the genus Ditylenchus. This finding was provided by molecular data written in the evolutionary variable, non-coding internal transcribed spacers (ITS1 and ITS2) of the ribosomal DNA (rDNA). In the current paper, we analyze the nucleotide sequences and predict the secondary structures of two expansion segments (D2, D3) of the 28S ribosomal RNA (rRNA)-coding gene for the plant-parasitic nematodes from the genus Ditylenchus and their related anguinids. In general, the expansion segment D2 appeared to be more variable than the segment D3 illustrating their different evolutionary constraints. Comparative analysis of the aligned sequences and predicted secondary structures revealed similar trend showing the tight relationships between the stem nematodes (D. dipsaci, D. gigas, D. weischeri) and gall-forming nematodes from the subfamily Anguininae. Phylogeny reconstructions disjoined the family Anguinidae into two monophyletic clusters (Clade 1 and 2). Clade 1 constitutes the stem nematodes (D. dipsaci, D. gigas, etc) and gall-forming nematodes from the genera Anguina, Heteroanguina, Subanguina and Mesoanguina, while clade 2 includes other Ditylenchus species like D. destructor and D. halictus. Collectively, deciphering the exact phylogenetic relationships within the family Anguinidae (Nematoda: Tylenchida) with respect to our results should provide a framework for a taxonomic revision in order to reflect biological history of these nematodes. In addition, we provide novel molecular data, which may be exploited in diagnostic tools for phytosanitary control of these economically important plant parasites.