The present study was designed to optimize a PCR-RFLP protocol for the molecular detection of Anaplasma sp. and to compare its prevalence in blood samples of equines from Southern Punjab (Pakistan) and to find out the risk factors involved in the spread of anaplasmosis. A total of 210 blood samples were collected from equines from 2 sampling sites (Dera Ghazi Khan and Khanewal districts). Data on the animals’ characteristics (age, species and gender) were collected through survey. PCR amplified the 577bp product specific for 16S rRNA gene of Anaplasma spp. in 9 blood samples (4.3% of total), [Dera Ghazi Khan (N = 3) and Khanewal (N = 6)]. These Anaplasma spp. positive blood samples were used for PCR amplification using A. phagocytophilum specific primers and parasite was detected in all of them. Also it was revealed that the characteristics of the animals i.e. age, gender, species had no significant association with the presence of Anaplasma sp. Hematological parameters remained unaffected while lymphocyte count was significantly lowered in A. phagocytophilum positive samples.