Tropical theileriosis (TT) is a tick-borne disease of cattle caused by the apicomplexan parasite Theileria annulata. It poses a major threat to the health of imported exotic breeds and, to a lesser extent, native breeds which have a degree of resistance to the disease. The study aimed to determine the prevalence of T. annulata infection in cattle in the arid and semi-arid regions of Algeria using serological and molecular methods. Bovine blood was tested for the presence of T. annulata-specific antibodies using the immunofluorescent antibody test (IFAT) and for parasite DNA using a polymerase chain reaction (PCR) for the gene encoding the major merozoite/piroplasm surface protein (Tams1). One hundred and sixteen individuals were screened and overall prevalences of 50.0 +/- 4.6 % (IFAT) and 64.7 +/- 4.4 % (PCR) were determined. There was moderate agreement between IFAT and PCR tests for the diagnosis of TT, with a sensitivity of 74.6 % and a specificity of 95.1 % for IFAT when compared against the PCR. Potential risk factors identified in this study were breed (OR = 2.25, 95% CI: 1.07-5.07), season of sampling (OR = 4.29, 95% CI: 1.97-10.9) and breeding system (OR = 8.60, 95% CI: 1.41-57.6). Sequential analysis of Tams1 PCR amplicons revealed three new haplotypes with 97.5-100% identity to each other and, overall, they were found to be similar to previously sequenced isolates from north African-south European lineage.