Maize (Zea mays L.) is a global food staple and is at risk from infection by the pathogenic fungus Aspergillus flavus L. The ubiquitous, soil-borne fungus causes ear rot of maize and produces the carcinogenic secondary metabolite known as aflatoxin. Aflatoxin B1 is the most potent carcinogenic mycotoxin known, causing hepatocellular carcinoma, along with many other serious health problems such as immunosuppression. Previous studies have shown that maize cob tissue plays an essential role in both facilitating and limiting the spread of the fungal pathogen A. flavus, however, little attention in the literature has been given to the cob. To date, there have not been any studies published describing the metabolome of maize cob tissue. This study assessed three different methods for disruption of maize cob tissue and investigated the global metabolome of maize cob of two resistant (Mp313E and Mp420) and two susceptible (B73 and SC212m) genotypes via liquid chromatography-mass spectrometry. Three treatments (control, water-inoculated, and fungus-inoculated) and three-time points (3, 9, 15 days after inoculation) were included in the experimental design. For the first time in maize cob, 69 metabolites were identified via the mzCloud online database. Out of them, 28 metabolites showed statistically significant differences in abundance across the treatments. Twenty-two metabolites were identified via Fragment Ion Search, of which 14 were statistically significant differences in abundance across the treatments. The majority of the metabolites identified where from the phenylpropanoid, linoleic acid, and terpenoid biosynthesis pathways. Thousands of unknown ions were detected, and for 521 compounds the formula could be derived, based on accurate monoisotopic masses. In the targeted metabolomics analysis, the MS3 spectral tree was obtained for zealexin B1 for the first time via a highly induced sample and was subsequently used to identify zealexin B1 in maize cobs (Va35). To date, this work is the sole metabolomic profiling study of maize cob tissue, and it provides insight into constitutive and induced molecular antifungal defenses of resistant and susceptible genotypes. A list of significant fungal-induced metabolites related to the maize-A. flavus defense response was compiled for further targeted metabolomic identification.