In several areas of Spain, the greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and the sweet potato whitefly, Bemisia tabaci (Gennadius), coexist in tomato, Lycopersicon esculentum Miller. For integrated pest management decision-making, it is important to know the abundance of each species, because they exhibit different abilities to transmit viruses, are susceptible to different biological control agents, and have different responses to insecticides. This study was conducted to provide information on the vertical distribution of T. vaporariorum and B. tabaci in tomato plants grown in greenhouses in winter and to determine the optimal sampling unit and the sample size for estimating egg and nymphal densities of both whitefly species. Eggs of T. vaporariorum were mainly located on the top stratum of the plant, whereas B. tabaci eggs were mainly found on the middle stratum. Nymphs of both species mainly concentrated in the bottom stratum of the plant. When pest abundance and low relative variation were considered, the bottom stratum was selected as the most convenient for sampling nymphs of both whitefly species. Conversely, the same two criteria indicated that either the top or the middle strata could be used when sampling T. vaporariorum and B. tabaci eggs. Several different sampling units were compared to optimize the estimation of nymphal and egg densities in terms of cost efficiency. One disk (1.15 cm in diameter) per leaflet collected from the top stratum of the tomato plant was the most efficient sampling unit for simultaneously estimating the egg densities of the two whitefly species.