Here we report the viability of natural chromosomal O-inversion structural homo- and heterokaryotypes in a population of D. subobscura from Avala Mountain, Serbia, subjected to cold and heat stress during spring 2004. These results were compared with those observed in the same population in September 2004. We detected protection of a great number of lethal genes in O-chromosome lethal heterozygous combinations, independently of inversion backgrounds and temperature stress. The positive epistatic interactions of mildly-deleterious genes observed only in O-inversion heterokaryotypes (at distinct temperatures) and lethal genes in O-inversion homokaryotypes (under temperature stress only) could be responsible for reducing the genetic load. The seasonal fluctuations in frequencies of several chromosomal arrangements and karyotypes indicate a seasonal adaptation to changeable environmental conditions. The sample of D. subobscura collected in spring 2004 was tolerant to cold but not to heat, and these results point to tolerance to extreme low temperatures, possibly as a result of natural selection. Also, a non-random distribution of distinct combinations of viability classes among several O-inversion karyotypes (under both optimal conditions and heat stress) was observed. This observation could be taken as evidence of co-adaptation: different capacities of several O-inversion karyotypes with different combinations of genes to tolerate a range of temperatures. We conclude that frequency-dependent and supergene selection are balancing selection mechanisms and are responsible for the protection of chromosomal inversions in natural populations of D. subobscura.