During the last decade, DNA bar-code is proposed as a novel concept in the identification of different animal species. It seems that result of DNA barcoding, taxonomy and systematic, due to some overlaps, could be useful simultaneously for constructing a better system for species delimitation, biodiversity measurement and assess of evolutionary relationships exiting among various taxons. Plentiful research projects are underway to investigate the concept of DNA bar-code. Among them, nematodes are from the first subject organisms that have been used to evaluate the efficacy of DNA barcoding. Entomopathogenic nematodes, including two families Heterorhabditidae and Steinernematidae are a noticeable group of these organisms. During the last decade, this group of nematodes has found an increasing importance in biological pest control programs. The important part of this progress is beholden to increase the information about species of these families that originate from developments in new array techniques. In the past few years, description of more than half of 92 species of entomopathogenic nematode have been achieved by using molecular data. Although, in this way, different markers and gene regions have been examined, most common method is DNA sequences ITS and 28S genes. Most studies that have been carried out about description and introduction of new species or evaluate phylogenetic relationships in two type genus (Steinernema and Heterorhabditis) are conducted by using analysis of ITS sequence. In the case of Steinernema species, however, 28S may be more useful. In this paper, beside the explanation of the evolution process used as molecular markers in basic studies of entomopathogenic nematode, latest findings in this area with analytical approach as well interpreting the current challenges have been discussed

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