Microsat.zip folder contains the 25-loci genotypes for the greater bee fly ( Bombylius major , N=591) and the ashy mining bee ( Andrena cineraria , N=182) in Luxembourg. For A. cineraria , two different genotyping datasets were produced; the first defined alleles based on the length of sequences (length-only data, LO), and the second defined allelic variants in terms of the occurrence of SNPs within each length class (sequence-information data, SI). Data are provided in genpop format, the first population corresponds to the rural sample, the second to the urban sample. Sample coordinates are provided in separate files. The SIMUPOP.zip folder contains the python and unix scripts employed to run the SIMUPOP simulations. These scripts were originally developed by Dr Diane Bailleul and subsequently modified by Dr Yacine Ben Chehida. Here, only small modifications and corrections were made to adapt the simulations to the study system of the present study. The data were analysed in the context of the article: " Genetic connectivity is maintained in two insect pollinators across a human-altered landscape " published in Insect Conservation and Diversity.