Gray leaf spot (GLS) caused by Cercospora zeae-maydis and C. zeina is an extremely devastating leaf disease that limits maize production annually. The use of GLS-resistant maize hybrids is the most cost-effective approach for reducing losses. Resistance to GLS is quantitatively inherited in maize (Zea mays L.) and further sources of resistance remain to be analyzed. Here, we detected qRgls1.06, a major quantitative trait locus for GLS resistance in bin 1.06 that explained approximately 55％of the phenotype variance. Fine mapping over 2 consecutive years localized qRgls1.06 to a 2.38-Mb region. Homozygous qRgls1.06WGR/WGR plants in DZ01 background displayed higher GLS resistance and 100-grain weight than DZ01 plants. The GLS responses of several susceptible elite inbred lines were improved by the introduction of qRgls1.06 by marker-assisted backcrossing. Our findings extend the understanding of the genetic basis of resistance to GLS and provide a set of resistant germplasm for genetic improvement of resistance to GLS in maize.