Introduction. Fruit flies (Diptera, Tephritidae) are among the major constraints in commercial horticulture in many African developing countries. Knowledge of the tephritid spectrum in any given area is a prerequisite for the development of an IPM program to alleviate the pest problem. We studied the fruit fly diversity in four main agro-ecological zones which are significant fruit-producing areas in the Morogoro Region, Tanzania. Materials and methods. Fruit fly diversity was investigated for one year (October 2004 – October 2005). Parapheromones, synthetic food attractant and protein-bait traps were used to trap the flies at the different locations in Morogoro region, Tanzania. One mixed orchard was selected at each of four locations representing the different agro-ecological zones of the region. Results. The recently introduced alien species, Bactrocera invadens, and three indigenous pest species, Ceratitis rosa, Dacus bivittatus and D. punctatifrons, were found at all the four sites, while Bactrocera cucurbitae, Ceratitis cosyra, Dacus chiwira and D. humeralis were found in three out of the four sites. The Sokoine University of Agriculture (SUA) Horticulture Unit and Mikese sites had the highest species diversity while the Mkindo and Nyandira sites had the lowest diversity. The between-habitats diversity (Beta diversity) was similar among the SUA, Mkindo and Mikese sites. Only the Nyandira site had a higher dissimilarity in comparison with the other three sites. Discussion. The most abundant species at low- and mid-elevation sites was Bactrocera invadens while, at high elevation, Ceratitis rosa was the dominant species. Protein-baited traps attracted the highest diversity of fruit flies in comparison with the more specific parapheromones. The synthetic food attractant (three-component lure) was less efficient compared with the protein bait. The large dissimilarity at the Nyandira site is probably due to the different types of host fruits found and grown in high altitude areas (mainly temperate fruits). Fruit fly species associated with these types of fruits, e.g., Ceratitis rubivora and Trirhithrum coffeae, were found only at this site.