Simple SummaryMycoplasma gallisepticumcauses respiratory disease in chickens. This study used RNA-seq to investigate the immune response of chicken embryos and chicks to infection. Weight loss and immune damage were observed in infected chickens of both ages. Transcriptome analysis revealed differentially expressed genes related to innate immunity and inflammation, with toll-like receptors and cytokine pathways playing a dominant role in the immune response. The immune response was stronger in embryos than in chicks. These findings provide valuable insights for developing disease control strategies.AbstractMycoplasma gallisepticum(MG) is a major cause of chronic respiratory diseases in chickens, with both horizontal and vertical transmission modes and varying degrees of impact on different ages. The innate immune response is crucial in resistingMGinfection. Therefore, this study aimed to investigate the innate immune response of chicken embryos and newly hatched chicks toMGinfection using comparative RNA-seq analysis. We found thatMGinfection caused weight loss and immune damage in both chicken embryos and chicks. Transcriptome sequencing analysis revealed that infected chicken embryos had a stronger immune response than chicks, as evidenced by the higher number of differentially expressed genes associated with innate immunity and inflammation. Toll-like receptor and cytokine-mediated pathways were the primary immune response pathways in both embryos and chicks. Furthermore, TLR7 signaling may play an essential role in the innate immune response toMGinfection. Overall, this study sheds light on the development of innate immunity toMGinfection in chickens and can help in devising disease control strategies.