Serve is the most important stroke in modern tennis, but the number of studies on professional women’s tennis is scarce. The aim of the present study was to identify differences in service direction and effectiveness regarding players’ ranking position; as well as to observe the differences according to the opponent’s laterality (right-handed or left-handed). A total of 108 matches from the US Open and Wimbledon tournaments were analyzed. Results showed a larger variability in service tactics in Wimbledon compared to the US Open. In addition, the direction and effectiveness of the service of right-handed players differed depending on the opponent’s laterality, especially against left-handed players. These results contribute to the knowledge of the service dynamics and performance in professional women’s tennis players.