In the present study, we report the sympatric occurrence of Ixodes ricinus (Linnaeus, 1758) and Ixodes inopinatus (Estrada-Peña, Nava, and Petney, 2014) in Tunisia. In total, 173 adult Ixodes ticks were collected from four sites (El Jouza, Tamra, Aïn Soltan, and Jbel Zaghouan) between February and April 2017, a period corresponding to the peak of activity of I. ricinus in North Africa. The morphological characters corresponded to both species; thus, we generated a total of 28 16S rRNA sequences and compared them with previously published data in GenBank. The two species were sympatric in Tamra, Aïn Soltan, and El Jouza, whereas collections in Jbel Zaghouan only yielded I. inopinatus. These results indicate that the two taxa are widespread in the humid area of northern Tunisia. The one tick collected in Jbel Zaghouan suggests that the distribution of at least I. inopinatus might extend to the sub-humid area. More studies are needed to fully comprehend the systematic status of the two taxonomic entities using multiple molecular markers and morphological characters; integrating these two identification methods are a necessary step toward a better understanding of the ecology and epidemiology of tick-borne diseases in Tunisia.