Mother birds can enhance growth and/or survival of chicks by supplying energetic and hormonal resources to the egg yolk (i.e. maternal effect). In several bird species, mothers supply higher level of testosterone to the eggs laid later within the clutch and enhance the growth of the chick to compensate for the disadvantage of the later hatching. As same as this within-clutch mechanism, mothers breeding later in the season can be expected to supply higher level of testosterone to the clutch. However, among-clutch seasonal variation in yolk testosterone levels has been rarely described. Here, we investigated among-clutch seasonal variation in the yolk testosterone level in Black-tailed Gulls Larus crassirostris, as well as within-clutch variation. Mean yolk testosterone level of eggs laid later within the clutch (“b-eggs”) was significantly higher than that of eggs laid earlier (“a-eggs”). The yolk testosterone levels of both a- and b-eggs of later breeders were significantly higher than those of earlier ones, while this trend was not observed in mean egg mass. Our results indicate that mothers establishing the clutch later in the season would invest more maternal testosterone into the clutch.