We assessed the efficacy of tetracycline, calcein and strontium chloride for validating the periodicity of otolith growth-increments in eight species of tropical marine gobies (family Gobiidae). We compared the number of fishes in which the otoliths were successfully marked when each of these chemicals was administered by intraperitoneal injection or immersion bath at a range of doses and immersion times. All three chemicals could produce a detectable mark in the otoliths of five reef-flat gobies, Asterropteryx semipunctatus, Amblygobius bynoensis, Istigobius goldmanni, Valenciennea muralis and Amblygobius phalaena; however, tetracycline injection at 50 mg kg−1 is recommended because it produced a brighter otolith mark than calcein and is cheaper and quicker to detect than strontium chloride. Calcein immersion treatment of 125 mg l−1 for 24 h was the most successful treatment for two estuarine gobiids, Favonigobius reichei and Glossogobius biocellatus. No treatment produced a detectable mark in the otoliths of the coral-dwelling goby Gobiodon histrio. Adequate care in the preparation of otoliths was found to be essential for detection of the validation mark produced by tetracycline and calcein. Otolith growth-increments were deposited daily in the seven species of goby for which validation was possible.