Nylon and maleic anhydride (MAH)-grafted polyolefin-based thin co-extruded multilayer films are widely used in packaging applications encountered in daily life. The molecular structure of the nylon/MAH-grafted polyolefin buried interface and molecular bonding between these two chemically dissimilar layers are thought to play an important role in achieving packaging structures with good adhesion. Here, the molecular bonds present at a nylon/maleic anhydride (MAH)-grafted polyethylene buried interface were systematically examined in situ for the first time using sum frequency generation (SFG) vibrational spectroscopy. The carbonyl stretching frequency region of the SFG spectra of a nylon/MAH-grafted polyethylene buried interface showed the presence of hydrolyzed MAH groups grafted to the polyethylene chain and very low levels of unreacted MAH enriched at the buried interface. The ability of SFG to detect these molecular species at the buried interface yields important understanding of the interfacial molecular structure and provides the basis for subsequent in situ studies of the bonding reaction between the grafted MAH and nylon directly at the interface. This understanding may guide the design of multilayer films with improved properties such as enhanced adhesion between polymer layers. The approach used in this study is general and is applicable to study the molecular characteristics of other buried interfaces of significance, such as buried interfaces involving polymers in solar cells, polymer semiconductors, and batteries. Nylon impact modification is another area of interest where the interaction between the MAH-grafted elastomer and the continuous phase of nylon is important.