The glass eel stage of the American Eel Anguilla rostrata marks the onset of the catadromous migration into estuarine or freshwater habitats, and the endocrine mechanisms underlying this habitat selection are still not well understood. Using a candidate genes approach, the aim of this study was to test for different patterns of gene expression related to (1) salinity preferences and/or (2) capture site to predict physiological differences between migratory behaviors. We performed analyses revealing the expression of genes coding for key hormonal factors or their receptors on glass eel‐stage American Eels collected at the mouths of three rivers on the east coast of Canada (Grande‐Rivière‐Blanche, St. Lawrence estuary; Rivière‐Saint‐Jean, Gaspé Peninsula; and the Mersey River, Nova Scotia); eels from the three systems displayed different salinity preferences (brackish water/salt water/freshwater) under laboratory conditions. Transcripts from genes coding for prolactin (PRL), thyroid‐stimulating hormone β subunit, type‐2 iodothyronine deiodinase (DIO‐2), thyroid hormone receptors αa and αb (THRαa and THRαb), growth hormone (GH), insulin‐like growth factor 1 (IGF‐1), and their respective receptors (GH‐R1 and IGF‐1R) were all detected in glass eels. No differences in the expression patterns were detected pertaining to salinity preference, but strong differences were found among rivers. Rivière‐Saint‐Jean glass eels, which were the longest and the least pigmented among the three rivers, were characterized by the highest expression of PRL, DIO‐2, and THRαb. Those from Grande‐Rivière‐Blanche showed an increase in IGF‐1R. Glass eels captured from these two rivers also exhibited the highest expression of GH and GH‐R1. Overall, these results confirm gene × environment interactions at the gene expression level when glass eels settle into their continental habitat. As such, our results also support the concept of the presence of different ecotypes in the Atlantic Canadian coast and in the estuary and Gulf of St. Lawrence.