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The interactions of lethal and non-lethal genes and their contributions to the viability of Drosophila inversion karyotypes are not well understood. This is especially true under variable environmental conditions. Here we examine the viability of natural chromosomal O-inversion homo- and heterokaryotypes in a D. subobscura population from Avala Mountain, Serbia. The observations we report were performed at a range of temperatures over several years. The heterotic effect of O-lethal heterozygotes on viability was found to be independent of the effects of inversion backgrounds and temperature. Positive epistatic interactions of lethal, mildly deleterious (subvital) and quasinormal (normal) genes were found in O-inversions in heterokaryotypes but not in homokaryotypes. These interactions were independent of temperature. This finding could explain the limitation of the genetic load in D. subobscura populations. In the population analyzed, annual fluctuations in the frequencies of certain chromosomal arrangements, karyotypes and non-lethal chromosomes under cold-stress temperatures seemed to indicate a correlation between these polymorphisms and environmental conditions. Our results indicate that there is a response in tolerance to extreme temperatures that may be due to natural selection. The differences in mean viability between some O-inversion karyotypic combinations indicate that there are differences in their tolerance to variable temperatures. All our results suggest that both frequency-dependent and supergene selection are mechanisms that protect O-chromosomal inversions. Chromosomal inversions may be genetically differentiated and coadapted complexes in D. subobscura populations.