Unrepaired or misrepaired radiation damage in mammalian chromosomes can result in micronucleus formation at the first cell division. This represents loss of genomic information which may cause cell death. To improve our understanding of the mechanism of radiation-induced micronucleus formation, we characterized micronucleus ultrastructure and identified the origin of micronucleus DNA. Immunofluorescence microscopy showed that micronuclei were structurally similar to main nuclei since they contained nuclear lamins A and C and were encapsulated by a network of vimentin intermediate filaments. The contents of radiation-induced micronuclei were characterized using fluorescence in situ hybridization to probe for DNA originating from chromosomes 2, 7, 11 and 16. We postulated that if incorporation of DNA into micronuclei were random, then the probability of chromosomal DNA in micronuclei would be related to the target, i.e. chromosome size. Our results demonstrated that incorporation of DNA from smaller chromosomes (11 and 16) was not different from expected values but incorporation of DNA from the larger chromosomes (2 and 7) was significantly greater than expected. Not all chromosomes in the human genome, therefore, were equally susceptible to genomic loss by micronucleus encapsulation. In conclusion, radiation-induced micronuclei have similar structural characteristics to main nuclei, chromosome damage and/or repair after ionizing radiation may be non-random, and micronucleus formation may reflect this variability.