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Mycoplasma 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 resisting MG infection. Therefore, this study aimed to investigate the innate immune response of chicken embryos and newly hatched chicks to MG infection using comparative RNA-seq analysis. We found that MG infection 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 to MG infection. Overall, this study sheds light on the development of innate immunity to MG infection in chickens and can help in devising disease control strategies.