Two experiments were conducted to study the effects of thermal manipulation on heat shock protein 70 (HSP70) transcription and immune cell characteristics' in Pekin duck embryos. In experiments I and II, fertile duck eggs were incubated at either a standard (S; 37°C) or high (H; 38.0°C) temperature from embryonic day (ED) 1 to 10. At ED11, half the eggs incubated at 37.5°C or 38.0°C were either kept at 37.5°C (SS) or 38.0°C (HH) respectively, or moved to the opposite incubator/temperature of either 38.0°C (SH) or 37.5°C (HS). Beginning on ED 21, all eggs (embryos) were incubated at 37.5°C. In experiments I and II, the mean egg shell temperature (EST) of eggs incubated at 38.0°C was higher than that from 37.5°C at ED10 (P < 0.01). In both experiments, the yolk free wet embryo body weight (YFWEBW) of embryos from the HS treatment was increased while that in the HH treatments was decreased compared with the SS treatment at ED25 (P < 0.01). In experiment II, embryos from the HH treatment had increased HSP70 mRNA compared with the SS treatment in the liver, thymus, and bursa (P < 0.05). The thymus and bursa from embryos in the SH treatment had increased MHCI mRNA when compared with the SS treatment (P < 0.05). The thymus from HH and SH treatments had increased MHCII mRNA while the bursa from the HH and SH treatments had decreased MHCII mRNA when compared with the SS treatment (P < 0.05). The spleen and bursa from the HH treatment had increased IL-6, IFNγ, and IL-10 mRNA when compared with the SS treatment (P < 0.05). The thymus and spleen from embryos in the HH treatment had increased CD8+/CD4+ ratios and CD4+CD25+ cell percentages compared with the SS treatment (P < 0.05). In conclusion, embryos incubated at 0.5°C higher than the standard incubation temperature from ED1 to 21 had increased HSP70 mRNA, MHCI, and inflammatory cytokines, and the immune response was skewed toward cell-mediated immunity.