Dissolved organic carbon (DOC) plays an important role in both abiotic and biotic processes within aquatic ecosystems. DOC is often defined as: allochthonous (terrestrially-derived), and/or autochthonous (within the water body), dominant components generally classified as humic acids, fulvic acids and/or proteins. DOC from different waterways also differ in their aromaticity, and molecular weight. This study collected and characterised DOC from various climatic regions and freshwater types (circumneutral; naturally acidic; lentic and lotic waters) around Australia to investigate differences in DOC composition among freshwaters. DOC was characterised using optical methods: absorbance and fluorescence spectroscopy. Excitation emission scans followed by parallel factor analysis (PARAFAC) showed that Australian DOC was characterised by three main components: protein-like, fulvic-like and humic-like compounds. DOC quality varied between circumneutral and naturally acidic waters, and circumneutral waters were further categorised by climatic zones (temperate versus tropical). There was no observed difference in DOC quality between lentic and lotic systems. Naturally acidic sites were dominated by highly aromatic (as indicated by the specific absorbance co-efficient at 340 nm: SAC340 (32 - 53 cm2 mg-1) and the specific UV absorbance co-efficient: SUVA254 (3.9 - 5.8 mg C−1 m−1)) allochthonous humic-like DOC of high molecular weight (as indicated by abs254/365 (3.6 - 4.2)). In contrast, the circumneutral waters were dominated by more autochthonous fulvic-like DOC of lower aromaticity and molecular weight (SAC340: 4 - 20 cm2 mg-1 and SUVA254: 0.8 – 2.9 mg C−1 m−1; abs254/365: 4.9 – 7.8). Tropical circumneutral sites were characterised by DOC higher in protein-like components, with lower aromaticity and molecular weight than DOC from circumneutral temperate sites.