High ambient temperature is one of the most important environmental factors negatively impacting poultry production and health. Genetics is an important contributor in mitigating the stress response to heat. Two genetically distinct highly inbred lines of similar body size (Leghorn and Fayoumi) were characterized for phenotypic differences in response to heat. At 14 days of age, birds were exposed to 38°C with 50% humidity for 4 hours, then 35°C until the conclusion of the experiment. Non-treated individuals were kept at 29.4°C for the first week and then 25°C throughout the experiment. Birds in the heat-stress group were inoculated at day (d) 21 with Newcastle disease virus (NDV) La Sota strain to investigate the effects of heat stress and NDV infection. Thirteen blood parameters were measured using the iSTAT blood analyzer at three stages: 4 h, 6 d, and 9 d post heat-stress treatment, representing acute heat (AH) exposure, chronic heat (CH1) exposure, and chronic heat exposure after virus infection (CH2), respectively. Most blood parameters were significantly changed with heat stress in Leghorns at AH and in Fayoumis at CH1 and CH2. The Leghorn line had significant acute responses with disrupted acid-base balance and metabolic disorders. The heat-resilient Fayoumis maintained a relatively well-balanced acid-base balance. The current study provides the comprehensive profile of biomarker signatures in blood associated with heat tolerance and suggests that PO2, TCO2, HCO3, and base excess can be served as potential biomarkers that can be used to genetically improve heat tolerance in poultry.