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<i>Fusarium</i> infections in humans (fusariosis) and in economically important plants involve species of several <i>Fusarium</i> species complexes. Species of the <i>Fusarium solani</i> species complex (FSSC) are the most frequent cause of human fusariosis. The FSSC comprises more than 60 closely related species that can be separated into three major clades by multi-locus sequence typing (MLST) using translation elongation factor 1-alpha (<i>TEF1-α</i>) and RNA polymerase II (<i>RPB2</i>) DNA sequences. The MLST nomenclature for clade 3 of the FSSC assigns numbers to species types (e.g., FSSC 2) and lowercase letters to identify unique haplotypes. The aim of this study was to analyse the genotypic and phenotypic characteristics of 15 environmental and 15 clinical FSSC isolates from Malaysia. MLST was used for the genotypic characterisation of FSSC isolates from various locations within Malaysia, which was complemented by their morphological characterisation on potato dextrose and carnation leaf agar. MLST identified eight different FSSC species: thirteen <i>Fusarium keratoplasticum</i> (i.e., FSSC 2), six <i>Fusarium suttonianum</i> (FSSC 20), five <i>Fusarium falciforme</i> (FSSC 3+4), two <i>Fusarium cyanescens</i> (FSSC 27)<i>,</i> and one each of <i>Fusarium petroliphilum</i> (FSSC 1), <i>Fusarium waltergamsii</i> (FSSC 7), <i>Fusarium</i> sp. (FSSC 12), and <i>Fusarium striatum</i> (FSSC 21). Consistent with previous reports from Malaysia, most (11 of 15) clinical FSSC isolates were <i>F. keratoplasticum</i> and the majority (9 of 15) of environmental isolates were <i>F. suttonianum</i> (5) or <i>F. falciforme</i> (4) strains. The taxonomic relationships of the isolates were resolved phylogenetically. The eight <i>Fusarium</i> species also showed distinct morphological characteristics, but these were less clearly defined and reached across species boundaries. Although <i>TEF1-α</i> and <i>RPB2</i> sequences were sufficient for the species identification of most FSSC isolates, a more precise MLST scheme needs to be established to reliably assign individual isolates of the species-rich FSSC to their geographically-, epidemiologically-, and host-associated sub-lineages.