In the past years, research has shown that the gut microbiome of insects has a catalytic role in improving the fitness of their host. This is no different in tephritid fruit flies, for which research has discovered and identified a wide variety of gut microbes in recent studies. This study has the goal of providing base line data on the level of variability of gut microbial assemblages of colony reared versus wild C. capitata as obtained by using different sample preparation protocols. We used bacterial 16S rRNA gene amplicon paired-end sequencing (MiSeq Illumina platform) to characterise and compare the gut bacterial composition of samples of colony and wild populations of C. capitata. Larval samples of colony populations, provided by the Insect Pest Control Laboratory of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, International Atomic Energy Agency (IAEA), included one long established (>30 years; > 400 generations) strain of Greek origin, one intermediate established (8 generation) strain of Australian origin and one newly (1 generation) established colony of Argentinian origin. Larvae from a wild population were collected in Italy in summer 2016. Individual larvae were processed by extracting bacterial DNA either from dissected guts or from whole larval bodies. Three replicates, consisting of five pooled individuals were considered for each combination of population (Greece, Australia, Argentina, Italy) and sample preparation protocol (bacterial DNA extraction from dissected gut or from whole larval body). Two- ways Permutational Multivariate Analysis of Variance (PERMANOVA) and Permutational Multivariate Analysis of Dispersion (PERMDISP) were used to test differences in gut microbiome composition and diversity. PERMANOVA showed high variability in the gut microbial assemblage composition of colonies of different origin and age. Significant variations in assemblage diversity were detected by PERMDISP. Conversely, no significant differences were observed between microbial assemblages obtained from dissected and not-dissected vouchers. These results will contribute to a better understanding of relationships between colony rearing protocols and fruit fly gut microbial assemblage diversity and composition.