ABSTRACTThis study characterises basic seminal and sperm parameters of puye (Galaxias maculatus maculatus), including sperm concentration, seminal plasma osmolality and cell viability. In addition, it provides a detailed description of sperm ultrastructure. Wild males (n = 21) were captured in southern Chile. A small and variable volume of milt was collected per male; the samples exhibited a high sperm concentration (55.84 ± 8.25 × 109 spermatozoa mL−1) and high viability (94.38% ± 2.78%). Spermatozoa had a total length of 26.9 ± 0.63 μm and consisted of an elongated head lacking an acrosome, a short mid‐piece with ring‐shaped mitochondrion, and a relatively short flagellum. The nucleus contained a deep nuclear fossa occupying almost the entire head, from which the flagellum emerged. The flagellum exhibits the classical 9 + 2 axoneme structure and consists of a long main piece and a short end‐piece. The main piece presents a pair of side‐fins of variable length, oriented in the plane of the central microtubules. This study provides the first detailed description of sperm ultrastructure in G. maculatus, revealing similarities with various fish families. This information could be valuable for future research on reproductive physiology, sperm evolution and potential applications in assisted reproduction and conservation.