The opague2 (o2) gene that alters the protein composition of maize (Zea mays L.) endosperm also influences starch digestibility for ruminants. The softer, less-dense kernel texture of o2 grain also improves starch availability. Unfortunately, the softer kernels of o2 maize adversely affect agronomic performance so the Quality Protein Maize (QPM) project at the International Center for Maize and Wheat Improvement initiated a breeding program to improve o2 genotypes. The objective of our study was to identify genomic regions influencing starch digestibility using quantitative trait loci (QTL) analysis for a cross of hard by soft-endosperm inbred lines of maize. The Texas A & M University maize breeding program developed 136 recombinant inbred lines (RILs) spanning a wide range of kernel vitreousness from the cross B73 o2 x CML161. Both lines were homozygous for the o2 allele. For each RIL, a 2-g kernel sample was ground through a 6-mm screen and placed in dacron bags with 50μm pore size. These were inserted in rumen-cannulated Holstein cows in mid to late lactation that were used for the in situ evaluations of dry matter (DM) disappearance at 0 and 14 h. There were three replications for each sample. Composite interval mapping revealed significant QTLs on chromosomes 1, 4, 6, and 7 for 14-h DM disappearance. For 0-h DM disappearance, QTLs were detected on chromosomes 3, 6, and 7, and several overlapped with the 14-h QTLs, which indicated a particle size effect. Several QTLs were also detected for the difference between for 14-h and 0-h DM disappearance, and these were probably more closely associated with in situ ruminal starch degradability. The findings from this study aid in determining breeding strategies for improving starch digestibility for corn silage where the starch is the largest and most available energy source for ruminants.