Ticks are major vectors of several pathogens of veterinary and medical importance. Hyalomma scupense is a potential vector of Theileria annulata, the parasite responsible for tropical theileriosis in cattle. This disease negatively impacts livestock production and causes substantial economic losses. Our study investigates the molecular and phylogenetic profiles of Theileria spp. in Algeria. A total of 40 H. scupense ticks were collected from Medea, Algeria, and five samples tested positive for Theileria spp. based on their COXIII mitochondrial partial sequences. These positive ticks were further analyzed using partial mitochondrial COXI and SSU 12S genes. The results revealed two novel haplotypes for the H. scupense 12S gene and one new haplotype for the H. scupense COXI gene. For T. annulata, one haplotype was identified, previously reported in Algeria from cattle blood. Phylogenetic analysis confirmed the genetic diversity of the detected haplotypes, providing insights into the evolutionary relationships of these parasites and their vectors. To our knowledge, this study represents the first molecular characterization of T. annulata from its vector, H. scupense, in Algeria. These findings highlight the need for continued research into the transmission dynamics and pathogenic mechanisms of this parasite to better understand its epidemiology and develop effective control strategies to mitigate its impact on livestock health.