Simple SummaryPrebiotics such as seaweeds have been proposed as positive influencers on hen health and productivity, including their response to stressors such as heat. The present study was undertaken to evaluate the effects of 3% red seaweed Chondrus crispus and 0.5% brown seaweed Ascophyllum nodosum supplements on the heat stress responses in two genetic strains of laying hens, Lohmann LSL-Lite (White) and Lohmann Brown-Lite (Brown). The short-term seaweed diet had little effect on blood characteristics, but long-term feeding affected several blood chemistry values. The seaweed supplement reduced the feed intake and improved the feed/egg efficiency in the short term, although did not consistently affect the long-term performance. Heat stress affected the leukocyte count and some plasma chemistry parameters, particularly in the Brown layer strain. Our results showed better production, feed efficiency and resistance to heat stress in the White layer strain, with significant liver enzyme changes in alkaline phosphatase (ALP), alanine aminotransferase (ALT) and gamma-glutamyl transferase (GGT). More research on the biological effects of seaweed supplementation is necessary to better understand its impacts on health and performance.This study was planned to investigate the effects of seaweed supplementation, genetic strain, heat stress and their interactions on laying hen performances, blood chemistry and hematology. In a short-term trial, laying hens of the two genetic lines Lohman LSL-Lite (White) and Lohman Brown-Lite (Brown) were supplemented with Chondrus crispus (CC) at 3% for 21 days, while a control group was not. In a long-term trial, the same two strains were assigned to control (0%), 3% red seaweed Chondrus crispus (CC) or 0.5% brown seaweed Ascophyllum nodosum (AN)-supplemented diets for 41 weeks, concluding with a four-week control or heat-stress period. The White hens displayed higher egg production and a lower feed/egg ratio. The short-term inclusion of CC significantly reduced the feed intake, weight gain and feed/egg ratio. The long-term seaweed intake affected the plasma albumin and gamma-glutamyl transferase (GGT) (p < 0.05), and there were significant strain-heat stress interactions; heat stress in the Brown birds was associated with reduced protein, globulin and glucose and increased cholesterol and GGT levels and higher heterophil-to-lymphocyte (H/L) ratios (p < 0.05) in response to heat stress (p < 0.05). In conclusion, a long-term seaweed supplementation affected the plasma protein and enzyme profiles, yet had little effect on hen leukocyte counts and the overall performance.