Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection ofBabesia bigeminaandTrypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR forB. bigeminaandT. evansiamplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% forT. evansi,B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P≤0.005) as compared toT. evansi(P≤0.05) andB. bigemina(P≤0.01) among various districts under study. A very low prevalence ofT. evansi(0.73%) andB. bigemina(0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latentT. evansiandB. bigeminainfection in cattle and buffaloes. Haematological evaluation revealed marked pathology inB. bigeminainfected group and in dual infected group in contrast to that infected withT. evansialone.