Hysterothylacium species are perhaps the most abundant and diverse group of marine ascaridoids. Identification of larval stages at specific levels is very problematic. This study describes the occurrence of Hysterothylacium larvae parasitizing the peritoneal cavity and mesenteries of Saurida tumbil purchased from fish markets in Saudi Arabia. Fish were visually inspected for nematodes using micro- and macroscopic examinations. Nematodes were assigned to genus level based on morphology and identified at specific level by sequence analyses of 18S rRNA and COI genes. Morphological examination by light microscopy showed that worms identified as third-stage larvae (L3) belonged to genus Hysterothylacium Ward and Magath, 1917. COI sequences from host confirmed identity of host as S. tumbil as it resulted in identical sequences from S. tumbil in GenBank. Identity of L3 based on 18S rDNA sequences confirmed their identity as H. thalassini and showed high similarity to sequences in GenBank. A unique hitherto sequences of L3 related to COI region have been reported herein. This is the first record of Hysterothylacium L3-stage parasitizing lizardfish in Saudi Arabia. Therefore, this study represents the importance of a combination of morphological and molecular tools for taxonomy and systematics of ascaridoids at specific level and confirming its host identity.