1. The aim of this work was to determine the frequency of occurrence of Toxoplasma gondii and genetically analyse isolates from a number of avian hosts in the southwest of Iran (Khuzestan province). The frequency of T. gondii was determined in free-range chickens (Gallus domesticus), sparrows (Passer domesticus), pigeons (Columba livia) and starlings (Sturnus vulgaris). 2. Isolates obtained from Toxoplasma-infected birds were subjected to molecular typing by PCR-restriction fragment length polymorphism (RFLP) with sequence analysis of the GRA6 gene. 3. The results showed that 41 (16·5%) of 241 samples of avian tissue were infected with T. gondii. Sparrows were most frequently infected (17 out of 64). 4. Analysis of the GRA6 gene by PCR-RFLP and DNA sequencing revealed Type II and III T. gondii were the predominant lineage, accounting for 19·5% and 80·5% of the isolates, respectively. 5. It was concluded that the use of this PCR test facilitated the diagnosis of T. gondii in avian hosts and the GRA6 PCR-RFLP method clearly differentiated between the three different T. gondii lineages. This study showed a higher prevalence of type III compared with type II T. gondii in infected avian hosts in southwestern Iran.