Atlantic bluefin tuna (BFT; Thunnus thynnus) is a migrating species straddling the North Atlantic Ocean and Mediterranean Sea. It is assumed that this species is divided into a western and an eastern stock, which spawn in the Gulf of Mexico and the Mediterranean Sea, respectively. To learn more about the reproductive behavior of the eastern BFT stock, we tracked gonadal development in adult fish that were sampled between April and July during three consecutive years (2003–2005). Sampling campaigns were carried out using common fishing methods at selected locations within the Mediterranean Sea, namely Levantine Sea, Malta, and Balearic Islands. An additional sampling point, Barbate, was situated northwest of the Straits of Gibraltar along the Atlantic coast. Morphometric parameters such as the total body mass (MB) and the weights of the gonads (MG) were recorded, and the respective gonadosomatic index (GSI) values were calculated. The data collected revealed two important trends: (1) GSI values are higher in fish caught in the eastern rather than the western locations across the Mediterranean Sea, and (2) the GSI reaches maximum values between late May and early June in Levantine Sea (eastern Mediterranean Sea), and only 2 and 4 weeks later in the central (Malta) and western (Balearic Islands) locations, respectively. The advanced gonadal development in BFT correlates well with higher sea surface temperatures. Our findings also distinguish the northern Levantine Sea BFT population (mean MB 78.41 ± 4.13 kg), and the Barbate BFT population with the greatest MB (all fish sampled > 100 kg). These data reflect a situation in which the eastern Mediterranean basin may function as a habitat for young BFT, until they gain a larger MB and are able to move to the Atlantic Ocean. However, the existence of genetically discrete BFT populations in the Mediterranean Sea cannot be ruled out.