Hemotropic mycoplasmas (hemoplasmas) are obligate Gram-negative bacteria that target red blood cells, and infect a wide range of hosts including cats, dogs, domestic ruminants, pigs, rodents and humans. The present study was designed for the molecular detection of Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum in feline blood samples collected from various pet clinics in Pakistan, by Polymerase Chain Reaction (PCR), using 16S rDNA as the target sequence. Clinical and epidemiological data was collected in all animals included in the study. M. haemofelis and C. Mycoplasma haemominutum DNA was detected by PCR respectively in 6.8% (10/148) and in 18.2% (27/148) of cat blood samples. Of these, two animals were co-infected with both agents. Sequencing and phylogenetic analysis was performed in M. haemofelis infected samples. Analysis of risk factors revealed that risk of M. haemofelis and of C. Mycoplasma haemominutum infection was significantly higher during summer months than during the winter season (P ≤ 0.01 in both agents). Cats older than one year of age were significantly more predisposed to C. Mycoplasma haemominutum infection than younger cats (P ≤ 0.001), and fever manifestation was significantly associated with M. haemofelis infection (P ≤ 0.001) in enrolled subjects. In conclusion, this study represents the first report of molecular detection of M. haemofelis and C. Mycoplasma haemominutum in cats from Pakistan. Therefore, this agents should be considered in cats from Pakistan presented with compatible clinical signs, mainly those presented during summer season, and in adult cats and cats presented with fever.