A new species from the family Tylenchidae was recovered in the grasslands of Isfahan province, Iran, and is herein described based on morphological and molecular data. Ottolenchus isfahanicus n. sp. is mainly characterized by having a faintly annulated cuticle; elongated, slightly sigmoid amphidial apertures developed metacorpus with distinct valve under LM; vulva located at 69.472.3% of body length; large spermatheca about 2.75 times of corresponding body width; and elongated conoid tail with broadly rounded tip. SEM observations showed that the lip region is smooth; the amphidial apertures are elongated, slightly sigmoid slits; and the lateral field is a simple band. It is further characterized by 477-to-515-μm-long females with delicate 5.7-to-6.9-μm-long stylets with small, slightly posteriorly sloping knobs, as well as functional males, in the population. The new species closely resembles O. facultativus, but is separated from it based on morphological and molecular data. It was further morphologically compared with O. discrepans, O. fungivorus, and O. sinipersici. The phylogenetic relationships of the new species with other relevant genera and species were reconstructed using near-full-length sequences of small subunit and D2-D3 expansion segments of large subunit (SSU and LSU D2-D3). In the inferred SSU phylogeny, the newly generated sequence of Ottolenchus isfahanicus n. sp. formed a clade with two sequences of O. sinipersici and sequences assigned to O. facultativus and O. fungivorus. In the inferred LSU phylogeny, the three newly generated sequences of the new species and LSU sequences of O. sinipersici and O. discrepans formed a clade.