Abstract: Haemosporidian parasites are widespread among birds globally, with impacts ranging from severe disease to negligible effects, particularly in host species that have coevolved with their parasites. Despite their ecological importance, the diversity and prevalence of these parasites in the Afrotropical region remain poorly understood, especially in urban environments. Our study investigated the prevalence and diversity of haemosporidian parasites in Afrotropical avian hosts, focusing on differences between urban and non-urban habitats. We screened 95 birds from various species in Nigeria and investigated whether urbanization is associated with changes in the prevalence and richness of lineages of three haemosporidian parasites (Haemoproteus, Plasmodium, and Leucocytozoon). We found a haemosporidian prevalence of 36.8% with genus-specific differences between urban and non-urban habitats. The probability of Haemoproteus infection was higher in urban than non-urban habitats, but Plasmodium and Leucocytozoon did not differ between these habitats. Moreover, Haemoproteus lineages were exclusively found in urban habitats, while most Plasmodium lineages were restricted to non-urban habitats. Notably, we expanded the knowledge on diversity of haemosporidian lineages and avian hosts in the Afrotropics, with the first-ever record of hPYNJOC1 and pLUME2 lineages for the region, and the addition of new hosts for four Haemoproteus and two Plasmodium lineages. Our findings highlight the complexity of host-parasite relationships and the need for further research into the dynamics of haemosporidian parasites in Afrotropical avian hosts inhabiting diverse habitats. Overall, our study contributes to a better understanding of the prevalence, diversity, and distribution of haemosporidian parasites in the Afrotropics, emphasizing the importance of continued surveillance and monitoring to inform strategies for avian conservation and management.