Pooled-segregant whole-genome sequence analysis allows efficient QTL mapping of complex traits. Increasing the recombination frequency by random inbreeding of first generation segregants can enhance QTL mapping resolution. We now show that upon mapping of QTLs underlying high acetic acid tolerance in yeast, inbreeding not only narrows but also causes disappearance and emergence of QTLs. After screening for strains with unusually high acetic acid tolerance, the best strain fermented glucose similarly in the absence and presence of 0.8 % (v/v) acetic acid in semi-anaerobic, small-scale fermentations (YPD, pH 4.0). The industrial strain, Ethanol Red, was used as inferior control strain. QTL mapping using haploid derivatives and pooled-segregant whole-genome sequence analysis with 27 superior F1 segregants revealed two major QTLs. After inbreeding, 27 superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance of a major QTL, in which HAA1, a known regulator of high acetic acid tolerance, was identified as causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, VMA7, were identified as causative alleles in the other major, but narrowed-down QTL and in three newly appearing QTLs, respectively. This work shows for the first time that increasing the recombination frequency can reveal a different network of causative genes underlying a complex trait and that a single strain can thus harbour different sets of causative genes able to establish the same polygenic trait.