Reproductive isolation may emerge from genetic incompatibilities that disrupt hybrid viability or fertility. Although such incompatibilities are well-documented between species, the occurrence and mechanistic basis of such incompatibilities within species remain poorly understood. Identifying such epistatic genic interactions is essential for understanding how reproductive isolation originates and how incipient divergence is maintained despite ongoing gene flow. Here, we aim to identify and characterize the genetic basis of intraspecific incompatibility within Tetranychus urticae mites. In our lab, we took advantage of a wide panel of near-isogenic T. urticae lines to screen for incompatibilities through no-choice genetic crosses. Genetic crosses uncovered a near-isogenic line that exhibits strong incompatibility. Specifically, phenotypic screening showed substantial variation in F1 female sterility in crosses involving this line. Sterility was strongly dependent on cross directionality; crosses with othe T. urticae males resulted in variable but often strong penetrance of female hybrid sterility. The reciprocal crosses resulted in little to no sterility, indicating a pronounced asymmetry. To determine whether cytoplasmic factors contribute to female sterility, we introgressed three alternative mitochondria into the incompatible line via iterative backcrosses. Mitochondrial introgression did not restore female fertility. Indeed, the strength of female hybrid sterility did not change significantly, demonstrating that the incompatibility is likely driven by nuclear genetic factors. Using recombinant haploid F2 males from several parental lines, we further resolve the number of nuclear loci that underpin female sterility. Ongoing genetic work is suggesting that multiple loci contribute to female sterility in this line. Histological investigation of sterile and fertile hybrid females at different ages showed that female sterility is associated with an early termination of oogenesis. Our research demonstrates the presence of a strong nuclear-based intraspecific genetic incompatibility that can cause complete female sterility. Together, these findings provide a foundation for dissecting the molecular mechanisms of early-stage reproductive isolation within species.