Trichomonadida are flagellated protozoa that infect a wide range of vertebrate species, including poultry such as Gallus gallus domesticus. These pathogens hold significant veterinary importance due to their role in the emergence of diseases that can severely reduce productivity and cause substantial financial losses. Traditional methods for identifying these protozoa are limited by the subtle morphological variations between species. Molecular studies on Trichomonadida species in North African poultry are scarce. This study is the first to investigate the genetic diversity and phylogenetic relationships of Trichomonadida species in Tunisian poultry farms using a multilocus molecular approach (18S rRNA and α-actinin1), providing valuable insights into their epidemiology. Based on the analysis of 18S rRNA sequences, the Tunisian samples were divided into four main clades. The first clade, identified as H. meleagridis, was detected in caecal samples and genomic DNA of H. gallinarum nematodes from ten Tunisian localities. This clade was further subdivided into two distinct clusters, designated genotype 1 and genotype 2. Analysis of both nuclear markers revealed that all Tunisian haplotypes clustered with those from France, belonging to genotype 2, with a genetic distance of 4% (18S rRNA) between the two clusters. Furthermore, the analysis revealed the coexistence of H. meleagridis and P. wenrichi protozoa infecting H. gallinarum, suggesting a potential mixed infection. Additionally, other Trichomonadida species, including Simplicomonas sp. and T. gallinarum, were detected in the caecal samples. Phylogenetic analysis (ML) demonstrated a close genetic relationship between H. meleagridis and P. wenrichi, as well as between Simplicomonas sp. and the Monoceromonas-Tritrichomonas group. Both genotypes 1 and 2 of H. meleagridis exhibited a sister-group relationship with P. wenrichi, suggesting a shared evolutionary origin. This study represents the first investigation into the genetic diversity of Trichomonadida species in Tunisian poultry, highlighting the utility of the 18S rRNA locus for evaluating genetic variation and identifying potential mixed infections.