Maternally provisioned yolk hormones have been determined to play critical roles in development across vertebrate taxa. This study ascertained the presence and concentration of thyroid hormones triiodothyronine (T(3)) and thyroxine (T(4)) in the maternal serum and yolk of the developing placental viviparous shark Sphyrna tiburo from one site adjacent to Tampa Bay and another within Florida Bay, Florida, USA. The developmental profile of T(3) in yolk showed a steady increase from pre-ovulation to post-ovulation and peaked to its highest concentration during the pregnancy stage. There was an increase in the T(3)/T(4) ratio in yolk during the pregnancy stage which suggests a possible increase in the conversion of T(4) to T(3) within yolk, possible embryonic endogenous production, or passive uptake of T(3) from uterine fluids. Similar to the pattern seen in yolk, maternal serum T(3) concentrations tended to increase as development progressed. The concentration of T(3) and T(4) in yolk from Tampa Bay was consistently higher than in yolk from Florida Bay. The differences in the patterns of thyroid hormones from these two locations may explain previously reported differences in the rate of embryonic development in the two locations.