Hemotrophic mycoplasma (hemoplasmas) is a cell wall-less bacterium causing infectious anemia in animals. As data on hemoplasmas infecting cattle in Malaysia is scarce, specific polymerase chain reaction assays were used for detection of hemoplasmas from blood samples of cattle and ticks in this study. Hemoplasma DNA was detected in 69 (69.0%) of 100 cattle blood samples obtained from different breeds. A total of 50.0% of the cattle in this study were infected with only Mycoplasma wenyonii, while 2.0% were infected with only Candidatus Mycoplasma haemobos and 17% were infected with both species. Based on sequence analysis of the partial or nearly full length sequences of hemoplasma 16S rRNA gene, the presence of M. wenyonii and Candidatus M. haemobos was confirmed. Hemoplasmapositive cattle of less than three years appeared to have higher infection rate compared to other age groups. M. wenyonii was identified for the first time in approximately 30% of cattle ticks (Rhipicephalus microplus and Haemaphysalis sp.) in this study. This study presents the first molecular evidence of hemoplasmas in Malaysian cattle and ticks.