We sought to determine whether a strontium chloride injection could be used to create a transgenerational otolith mark in steelhead Oncorhynchus mykiss. Two strontium injection trials and a survey of strontium: calcium (Sr:Ca) ratios in juvenile steelhead from various steelhead hatcheries were conducted to test the feasibility of the technique. In both trials, progeny of fish injected with strontium had significantly higher Sr:Ca ratios in the primordial region of their otoliths, as measured by an electron wavelength dispersive microprobe. In trial 1, the 5,000-mg/L treatment level showed that 56.8% of the otoliths were correctly classified, 12.2% being misclassified as belonging to the 0-mg/L treatment. In trial 2, the 20,000-mg/L treatment level showed that 30.8% of the otoliths were correctly classified, 13.5% being misclassified as belonging to the 0-mg/L treatment. There were no differences in the fertilization rates of eggs or survival rates of fry between the treatment and control groups. The Sr:Ca ratios in otoliths collected from various hatchery populations of steelhead varied and were greater than those found in otoliths from control fish in both of our injection trials. This study suggests that the marking technique led to recognizable increases in Sr:Ca ratios in some otoliths collected from fry produced by injected females. Not all progeny showed such increases, however, suggesting that the method holds promise but requires further refinement to reduce variation. Overall, there was a correct classification of about 40% across all treatments and trials; the variation in Sr:Ca ratios found among experimental trials and hatcheries indicates that care must be taken if the technique is employed where fish from more than one hatchery could be involved.