Although the pan-genome concept originated in prokaryote genomics, an increasing number of eukaryote species pan-genomes have also been analysed. However, there is a relative lack of software intended for eukaryote pan-genome analysis compared to that available for prokaryotes. In a previous study, we analysed the pan-genomes of four model fungi with a computational pipeline that constructed pan-genomes using the synteny-dependent Pan-genome Ortholog Clustering Tool (PanOCT) approach. Here, we present a modified and improved version of that pipeline which we have called Pangloss. Pangloss can perform gene prediction for a set of genomes from a given species that the user provides, constructs and optionally refines a species pan-genome from that set using PanOCT, and can perform various functional characterisation and visualisation analyses of species pan-genome data. To demonstrate Pangloss’s capabilities, we constructed and analysed a species pan-genome for the oleaginous yeast Yarrowia lipolytica and also reconstructed a previously-published species pan-genome for the opportunistic respiratory pathogen Aspergillus fumigatus. Pangloss is implemented in Python, Perl and R and is freely available under an open source GPLv3 licence via GitHub.