Wildlife animals have integral significance in ecological and economic stability. They are becoming extinct. One of the key roots of endangerment is tick-borne diseases. Anaplasma, Babesia, and Theileria are major parasites transmitted by obligatory hematophagous arthropods known as ticks to wildlife, livestock, and humans. In the current study, the creation of novel primers and optimization of a multiplex PCR assay to detect hemoparasites were done. Blood samples were collected from a total of 60 captive Gazella bennettii, family Bovidae, and Axis porcinus, family Cervidae, from different wildlife parks and zoos in Punjab, Pakistan. Both families were targeted during sampling to increase the sample count and obtain a variety of ungulates. Microscopy was performed using the Giemsa staining method, and 40 samples tested positive, which were then proceeded with PCR. Total genomic DNA was extracted for molecular detection by PCR. This study comprised three sets of newly designed primers based on genus to optimize multiplex PCR for melting temperature and crosslinking among primers. The results of our multiplex PCR assay were completely consistent with monoplex PCR using newly designed primers. Results of multiplex PCR revealed the presence of total hemoparasitic species infection in 65% of samples, whereas individual infections of Anaplasma, Babesia, and Theileria were recorded as 25%, 0%, and 40%, respectively. PCR provided accurate results supported by sequencing. This multiplex PCR assay provides a valuable complementary tool in routine, simultaneous, early, and accurate detection of Anaplasma, Babesia, and Theileria genera that helps control antibiotic resistance and save endangered species. Wildlife animals have integral significance in ecological and economic stability. They are becoming extinct. One of the key roots of endangerment is tick-borne diseases. Anaplasma, Babesia, and Theileria are major parasites transmitted by obligatory hematophagous arthropods known as ticks to wildlife, livestock, and humans. In the current study, the creation of novel primers and optimization of a multiplex PCR assay to detect hemoparasites were done. Blood samples were collected from a total of 60 captive Gazella bennettii, family Bovidae, and Axis porcinus, family Cervidae, from different wildlife parks and zoos in Punjab, Pakistan. Both families were targeted during sampling to increase the sample count and obtain a variety of ungulates. Microscopy was performed using the Giemsa staining method, and 40 samples tested positive, which were then proceeded with PCR. Total genomic DNA was extracted for molecular detection by PCR. This study comprised three sets of newly designed primers based on genus to optimize multiplex PCR for melting temperature and crosslinking among primers. The results of our multiplex PCR assay were completely consistent with monoplex PCR using newly designed primers. Results of multiplex PCR revealed the presence of total hemoparasitic species infection in 65% of samples, whereas individual infections of Anaplasma, Babesia, and Theileria were recorded as 25%, 0%, and 40%, respectively. PCR provided accurate results supported by sequencing. This multiplex PCR assay provides a valuable complementary tool in routine, simultaneous, early, and accurate detection of Anaplasma, Babesia, and Theileria genera that helps control antibiotic resistance and save endangered species.