Molecular dating estimates the origin of the fungal clade to the Pre-Cambrian. Yet, the oldest unambiguous fungal fossils date to the Ordovician and show remarkable diversity and organizational development. Recent studies have suggested that the dates for the emergence of fungi in the fossil record may be pushed back to the Proterozoic. However, the nonspecificity of the methods used in those studies necessitates the employment of a wider variety of analytical techniques that can independently verify the presence of chitin, a crucial prerequisite in the assignment of fungal affinity, particularly of putative fossils from the Pre-Cambrian. In this paper, we propose Py-GC × GC-TOFMS as an example of one such technique. We analyze fungal fossils from the Pliocene. We find that a suite of N-bearing compounds are present in the pyrolysis products of these fossils, from which we suggest that 3-acetamidopyrones and their methylated homologues can serve as specific pyrolytic markers for chitin. We discuss both how this technique can potentially be used to differentiate between biopolymers, including those similar to chitin such as peptidoglycan, and the potential implications of identifying such markers in fossils from deep time. We conclude that Py-GC × GC-TOFMS is a promising technique that can potentially be used alongside, or independent of, staining methods to detect the presence of chitin in fossils.