Tsetse flies (Diptera: Glossinidae) are the main vectors of Human African Trypanosomiasis (HAT), or sleeping sickness and Animal African Trypanosomiasis, (AAT) or Nagana in Sub Saharan Africa. In Ghana, whilst HAT is no longer a major public health issue, AAT is still widely reported and causes considerable losses in the livestock sector resulting in major impacts on agricultural production, livelihoods and food security in the country. Application of morphometric techniques can reveal the existing level of population differentiation in tsetse flies, providing guidance on the distribution of genetically defined subpopulations. Morphometric techniques were used to compare size and shape of three tsetse fly species- G. m. morsitans, G. p. palpalis and G. tachinoides of Ghana, and also compare populations of G. p. palpalis collected from three geographical regions (Northern, Eastern and Western) of Ghana. Flies were sampled from four sites in the Western, one site in the Eastern and three sites in the Northern Region using standard un-baited biconical traps. Right wings and right hind legs of selected flies from different collection sites were removed and mounted on microscope slides using glycerin as the mounting medium. Images of the prepared slides were captured under a Leica EZ4 D microscope with an inbuilt camera connected to a laptop. Linear and proportions of wing and hind tibia measurements were arcsine-root transformed before analyzing with a general linear model in analysis of variance (ANOVA). Multivariate statistical analyses were used to detect any possible variations. Results of the GLM analyses of linear and ratio data revealed that different linear combinations can be used to characterize tsetse species of different populations. The ratio value hind tibia/wing length (th/at) significantly distinguished fly populations into four groups, Northern, Eastern, Western and the lab colony; this is an indication that hind tibia/wing length is a good morphometric feature which can be used to discriminate flies from different regions of Ghana. The principal components and canonical variates as well as Mahalanobis squared distances confirmed linear and ratio separations. Therefore based on these differences in morphometric characters observed, the three tsetse species were distinguished from each other. Similar work on morphometrics needs to be done to include more regions and many other body parts such as proboscis length, antennal length, thorax and abdomen length and width in order to establish stronger morphometric tools for discriminating different tsetse fly species.