The Sand cat (Felis margarita) is a small-sized felid found in sand and stone deserts ranging from the north of Africa to Asia, with the Arabian Peninsula as its centre of distribution. The Sand cat captive breeding program at the Breeding Centre for Endangered Arabian Wildlife (BCEAW), Sharjah, UAE, has experienced high newborn mortality rates, and congenital toxoplasmosis was recently recognized as one of the causes of this mortality. In the present study, one 18-month-old Sand cat (FM019) died of acute toxoplasmosis-associated hepatitis and pneumonitis acquired after birth; Toxoplasma gondii was demonstrated in histological sections which reacted with T. gondii polyclonal antibodies by immunohistochemistry (IHC). T. gondii DNA was found by PCR of extracted DNA from liver and lung tissues of this cat. Antibodies to T. gondii were found in serum examined in 1:1600 dilution in the modified agglutination test (MAT); its 2-year-old cage mate seroconverted (MAT titer 1:3200) at the same time. Another Sand cat (FM017) was euthanized because of ill health when 3 years old; its MAT titer was >1:3200, and T. gondii tissue cysts were found in brain, heart, ocular muscles and skeletal muscle, confirmed by IHC. Viable T. gondii was isolated by bioassays in mice inoculated with tissues of another chronically infected Sand cat (FM002); T. gondii was not found in histological sections of this cat. T. gondii antibodies were found in several species of animals tested, notably in 49 of 57 wild felids at BCEAW. A 7-year-old Sand cat (3657) from Al Wabra Wildlife Preservation (AWWP), Doha, State of Qatar died of acute visceral toxoplasmosis with demonstrable T. gondii tachyzoites by IHC, and T. gondii DNA by PCR, and a MAT titer of >3200. T. gondii antibodies were found in 21 of 27 of wild felids at AWWP. PCR-RFLP genotyping at 10 genetic loci revealed that these T. gondii isolates from Sand cat (FM002 and FM019) at BCEAW have an atypical genotype, which was previously reported in T. gondii isolates of dogs from Sri Lanka. The genotype from the cat from AWWP (3657) is a genetic Type II strain with a Type I allele at locus Apico. This is the first report of genetic characterization of T. gondii isolates from Middle East.