The subfamily Hoplolaiminae included economically important plant parasitic nematodes and consisted of more than 400 species, all having the diagnostic characters of a strongly annulated cuticle and a large stylet. Among the Hoplolaiminae genera, the genus Hoplolaimus species include species such as H. columbus, and H. galeatus that cause serious damage to crops and turf grass in the Southeastern United States. Traditional identification of species has been approached by interspecific variation of phenotypic traits that rely on morphological and morphometric characters. However, these taxonomic criteria are sometimes not practical because of their limited ability to discriminate species among closely related groups due to overlapping of important taxonomic characters. The exact species identification is needed to control target nematode and also quarantine. Therefore, genetic studies for development of molecular diagnostics, population biology, and disease management are required. In recent years, many molecular diagnostic methods have been used for the identification of plant parasitic nematodes. Advanced molecular techniques have been used that test traditional identification methods. In our studies, Hoplolaimus species showed that high genetic divergence in rDNA sequence is combined with low morphological diversity. Based on genetic information, we developed multiplex PCR for H. columbus, H. galeatus, and H. magnistylus and successfully amplified mixed populations. In molecular phylogeny, the subfamily Hoplolaiminae is an important out‐group of the Heteroderidae, a notorious plant parasite nematode group. Molecular phylogeny of the Hoplolaiminae will help us understand of pathways of pathogenesis. In our phylogenetic analysis using D2 and D3 expansion segments of 28S gene, the molecular data supported morphological based taxonomic schemes. To reconstruct more reliable phylogenetic analysis, correct assignment of each nucleotide within multiple sequence alignment is an important step. Sequence alignments based on secondary structure have been proposed as new alternative methods to obtain this goal. We predicted the secondary structure of D2 and D3 domain using computational predictions method such as the minimization energy method and comparative sequence analysis (co‐variation). Predicted secondary structure included 18 species with two outgroup species, Globodera rostochiensis, Rotylenchulus reniformis. Consensus secondary structure was obtained from closely related and distantly related species. Phylogenetically informative characters were distributed in the stem region (86.7%). These results support the effectiveness of stem and loop regions for phylogenetic analysis of the Hoplolaiminae.