Anaplasma phagocytophilum (A. phagocytophilum) is a tick-borne rickettsial organism of zoonotic significance that has been alleged for frequent clinical infections in domestic animals including cats. The current study aimed to investigate the molecular prevalence of A. phagocytophilum in domestic cats. The study also focused on the assessment of possible risk factors and hematological alterations associated with this infection. For this purpose, 384 blood samples were first examined microscopically and then by PCR targeting the 16S rRNA gene. The samples found positive on PCR were purified and sequenced later on. The data for the evaluation of risk factors was recorded and statistically analyzed. The present study found 3.39% and 7.03% of cat samples positive for A. phagocytophilum on microscopy and PCR, respectively. The sequences of A. phagocytophilum presented high similarity with isolates from Thailand with relatively less similarity with isolates from India and China. Chi-square analysis revealed previous tick history, use of ectoparasiticide, housing type and grooming practices as potential risk factors (P<0.05) for disease dynamics. Comparative hematological analysis revealed that the number of leukocytes, erythrocytes, hemoglobin, hematocrit, and platelets was significantly decreased (P<0.05) in cats suffering from A. phagocytophilum compared to the healthy ones. To our knowledge, no study has been performed in the past to detect the presence of A. phagocytophilum in domestic cats of Pakistan. The current study may help in the adoption of adequate tick-control strategies for the control of A. phagocytophilum infection in domestic cats.