Anaplasmosis is caused by a Gram-negative obligate intracellular bacterium of the genus Anaplasma with the pathogen having a zoonotic impact. The study aimed to estimate the prevalence of anaplasmosis in Pakistan, to unravel the association of potential risk factors, and to investigate the effect on hematological parameters in affected small ruminants. A total of 150 (n = 75 sheep; n = 75 goats) blood samples were initially screened microscopically and then subjected to PCR targeting the amplification of the 16S rRNA gene fragment of Anaplasma. The PCR-based positive samples were then processed for sequencing. Statistical analysis regarding risk factors was performed using R software. The study revealed an overall 29.33% (44/150) prevalence of anaplasmosis in small ruminants. Sheep had higher (P > 0.05) prevalence (32%) as compared to goats (25.30%). The final statistical model resulting from backward elimination showed only tick infestation as a significant predictor of infection status. The phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed 9 study isolates clustered together and showed a close resemblance (99%) with Anaplasma ovis isolate (DQ837600) from Hungary. One of the isolates showed (99%) similarity with the isolate of Anaplasma marginale (MH155594) from Iraq. Furthermore, the hematological parameters pack cell volume, red blood cells, hemoglobin, white blood cells, granulocytes, monocytes, lymphocytes, and platelet count were decreased in Anaplasma-positive animals. This is the first study at the molecular level to characterize Anaplasma spp. in small ruminants of Pakistan, and it will be useful in developing control strategies for anaplasmosis.