Maize (Zea mays subsp. mays) yield loss from arthropod herbivory is substantial. While the basis of resistance to major insect herbivores has been comparatively well-studied in maize, less is known about resistance to spider mite herbivores, which are distantly related to insects and feed by a different mechanism. Two spider mites, the generalist Tetranychus urticae, and the grass-specialist Oligonychus pratensis, are notable pests of maize, especially during drought conditions. We assessed the resistance to both mite species of 38 highly diverse maize lines, including several previously reported to be resistant to one or the other mite species. We found that line B96, as well as its derivatives B49 and B75, were highly resistant to T. urticae. In contrast, neither these three lines, nor any others included in our study, were notably resistant to O. pratensis. Quantitative trait locus (QTL) mapping with F2 populations from crosses of B49, B75, and B96 to susceptible B73 identified a large-effect QTL on chromosome 6 as underlying T. urticae resistance in each line, with an additional QTL on chromosome 1 in B96. Genome sequencing and haplotype analyses identified B96 as the apparent sole source of resistance haplotypes. Our study identifies loci for use in maize breeding programs for T. urticae resistance, as well as to assess if the molecular-genetic basis of spider mite resistance is shared with insect pests of maize, as B96 is also among the most resistant known maize lines to several insects, including the European corn borer, Ostrinia nubilalis.