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Toll-like receptors (TLRs) are a critical family of pattern recognition receptors (PRRs) that are tightly regulated by complex mechanisms involving many molecules to ensure a good response to foreign invaders. In this study, we identified and analyzed the sequence characteristics and homology of four TLR genes (<i>tlr1</i>, <i>tlr5s</i>, <i>tlr5m</i>, and <i>tlr14</i>) from the swamp eel. Sequence similarity analysis, functional domain prediction, and phylogenetic analysis supported their annotation and orthologies. Their relative expression levels in different tissues were assessed. The four TLRs were expressed in all tested tissues. <i>tlr5m</i> was highly expressed in the spleen, whereas <i>tlr5s</i> was highly expressed in the kidney and spleen. High expression levels of <i>tlr1</i> and <i>tlr14</i> were detected in the spleen and muscle. These results indicated that the TLRs are related to host immunity. Moreover, the differential expression of TLRs was examined after <i>Aeromonas veronii</i> infection, which showed that all the TLR genes were induced with diverse patterns. <i>tlr1</i> was significantly downregulated in the spleen after <i>A. veronii</i> challenge. In the kidneys and intestines, <i>tlr1</i> expression decreased initially and then increased, with its lowest level at 4 h. <i>tlr5s</i> expression was upregulated significantly in three tissues at 1, 4, and 12 h, with the maximum expression at 1 h, indicating that <i>tlr5s</i> actively responded to bacterial invasion in the early stage of the challenge. <i>tlr5m</i> showed tissue specific expression: it was slightly upregulated in the intestines and spleen and downregulated in the kidneys. The expression pattern of <i>tlr14</i> was similar to that of <i>tlr5s</i>, and both reached maximum expression at 1 h after infection. Collectively, our results indicated that TLRs might play important roles in the innate immune response against Gram negative bacteria in the swamp eel.