Background: Members of the genus Ichthyophthirius are some of the most notable ectoparasitic ciliates affecting freshwater fishes globally. While infections have been documented in common carp (Cyprinus carpio) and goldfish (Carassius auratus) in Iraq, molecular confirmations and recent updates to host range data are lacking. Considering the recent expansion of aquaculture and the translocation of fish, the potential for greater dissemination of parasites among both native and introduced aquatic ecosystems is concerning. Aim: The primary goal of the current study was to molecularly identify and phylogenetically characterize Ichthyophthirius spp. affecting freshwater fishes in the Al-Diwaniyah River, Iraq, and examine new extensions of host range. Methods: A total of 720 fish specimens belonging to five species were collected and examined between October 2023 and October 2024. Skin and gill tissues were microscopically investigated for the presence of ectoparasitic infections. From these positive samples, DNA was extracted, and the 18S rRNA gene was amplified. Subsequently, the PCR products underwent sequencing, and the resulting data were analyzed using the BLAST and Maximum Likelihood (ML) methods for phylogenetic analysis. Results: Ichthyophthirius spp were found only in skin tissues, affirming strong predilection or specific tissue preference. In molecular amplification, clear 18S rRNA products were found within the range of 1500–3000 bp. In comparison to 18S rRNA sequences of Ichthyophthirius spp, analyses of the sequences showed 98–100% similarity to any of the global reference sequence of Ichthyophthirius in the GenBank. In the phylogenetic reconstruction, Iraqi isolates formed a distinct clade with considerable support of Ichthyophthirius, which is almost identical with Asian strains, albeit minor nucleotide variations which suggest lateral stability. Although the parasite has been documented in Cyprinus carpio and Carassius auratus, this study reports Ichthyophthirius spp. for the first time in Oreochromis aureus, Garra variabilis, and Oreochromis niloticus in Iraq. Conclusion: Molecular data in this study serve to confirm the presence of Ichthyophthirius spp. in the Iraqi freshwater system. This study also expands the number of documented hosts to several cyprinid and cichlid species. Findings of this study are important in the understanding of parasite ecology, transmission cycle, and the possible impacts of aquaculture of Iraq.