From 35 species of marine fishes (n = 327) from the South China Sea, 237 nematode larvae were collected and identified morphologically as Anisakis. Genomic DNA was isolated from each larva and subjected to PCR-based RFLP and targeted sequencing of a nuclear ribosomal DNA region between the 3-end of the small subunit and 5-end of the large subunit of the rRNA genes (= internal transcribed spacers, ITS+). Four different RFLP profile combinations (sets) were detected for all restriction endonucleases (HinfI, HhaI, and TaqI), of which three were characteristic of Anisakis typica, A. pegreffii, and A. physeteris, respectively. One profile set (for sample CA-2012) was linked to an ITS+ sequence that was identical to a previously published sequence of Anisakis sp. (sample HC-2005; originating from the African shelf) and another sequence (PH-2010; Madeira, Portugal). Phylogenetic analysis was carried out using the ITS+ sequence data from this study and reference sequences from the GenBank database. Neighbor joining and maximum parsimony trees displayed three clades. Clades I and II included nine described species of Anisakis, including all type I and type II larvae; clade III represented some undescribed species of Anisakis. Morphological comparison showed that Anisakis sp. CA-2012 was distinct from type I and type II larvae based on its tail shape and ratio of tail length to body length. The phylogenetic analysis and morphological characters suggest that Anisakis sp. CA-2012 represents a new record, now called Anisakis type III larvae.