Significant differences in softening rate have been reported between melting flesh in peach and nectarine varieties. This trait seems to be controlled by several genes. We aimed to identify candidate genes involved in fruit softening rate by integrating quantitative trait loci (QTL) and expression QTL (eQTL) analyses, comparing siblings with contrasting softening rates. We used a segregating population derived from nectarine cv. ‘Venus’ selfing, which was phenotyped for softening rate during three seasons. Six siblings with high (HSR) and six with low softening rate (LSR) were sequenced using RNA-Seq. A group of 5,041 differentially expressed genes was identified. Also, we found a QTL with a LOD (logarithm of odds) score of 9.7 on LG4 in all analyzed seasons. Furthermore, we detected 1,062 eQTLs, of which 133 were found co-localizing with the identified QTL. Gene Ontology (GO) analysis showed ‘Response to auxin’ as one the main over-represented categories. Our findings suggest over-expression of auxin biosynthetic related genes in the HSR group, which implies a higher expression and/or accumulation of auxin, thereby triggering fast softening. Conversely, the LSR phenotype might be explained by an altered auxin-homeostasis associated with low auxin levels. This work will contribute to unraveling the genetic mechanisms responsible for the softening rate in peaches and nectarines and lead to the development of molecular markers.