Exposure to high temperatures is known to impair immune functions and disease resistance of poultry. Characterizing changes in the transcriptome can help identify mechanisms by which immune tissues, such as the thymus, respond to heat stress. In this study, 22-day-old chickens from two genetic lines (a relatively resistant Fayoumi line and a more susceptible broiler line) were exposed to acute heat stress (35 °C) and/or immune simulation with lipopolysaccharide (LPS; 100 µg/kg). Transcriptome responses in the thymus were identified by RNA-sequencing (RNA-seq). Expression of most genes was unaffected by heat and/or LPS in the Fayoumi line, whereas these treatments had more impact in the broiler line. Comparisons between the broiler and Fayoumi transcriptomes identified a large number of significant genes both at homeostasis and in response to treatment. Functional analyses predicted that gene expression changes impact immune responses, apoptosis, cell activation, migration, and adhesion. In broilers, acute heat stress changed thymic expression responses to LPS and could impact thymocyte survival and trafficking, and thereby contribute to the negative effects of high temperatures on immune responses. Identification of these genes and pathways provides a foundation for testing targets to improve disease resistance in heat-stressed chickens.