Background: Mosquito colony populations often show significant changes in their population genetic make-up compared to the field populations that were used as founding source. Most of the changes that have been reported are indicators of depletion in the overall genetic diversity of the colony populations. The Sterile Insect Techniques programme of mosquito control that is underway in Northern Sudan uses sterilized males produced from a laboratory-maintained colony population. The genetic diversity of an advanced generation of this colony population was quantitatively assessed and compared to the field population from which the colony was derived. Methods: Anopheles arabiensis mosquito samples from the 13th generation of the colony, and from the locality that was the source of the first generation of the colony, were genotyped at 11 microsatellite loci distributed throughout the species’ genome. Standard population genetic analyses were carried out to quantify and compare their population genetic make-up and diversities. Results: The colony samples showed significant reduction in the total number of alleles, the numbers of rare and private alleles, and the fractions of heterozygote individuals at all the loci. The pattern of change is consistent with the expected effect of the use of a small number of mosquitoes when the colony was established. Departure from Hardy–Weinberg equilibrium in the direction of homozygote excess was observed at some loci and attributed to the presence of null-alleles. Conclusions: This study highlights the need for broad sampling when initiating colony populations and for ongoing assessment of the population genetic make-up of colony populations. Previous assessments of survivorship, dispersive behaviour and swarm formation indicate that the inbreeding and reduced genetic variability reported in this study may not have had direct fitness consequences yet. However, noting the lessons learned in other SIT programmes about the impact of colonization on male sexual behaviour and longevity, as well as other inbreeding related adverse effects, a systematic investigation of these potential effects is recommended because they have direct impact on the ultimate success of the programme.