Evidence exists that omega-3 long-chain polyunsaturated fatty acids (ω3 LC-PUFA, also termed LC omega-3 oils) are important in the prevention and treatment of a range of human diseases and in the development of neurological functions in infants.  Naturally occurring ω3 LC-PUFA are predominantly found in marine sources (e.g. algae and fish), with high concentrations occurring in oily fish species (e.g. tuna and salmon) through bio-accumulation up the food-chain.  Increased pressure on wild fish stocks has prompted extensive global research into generating new sources of these LC omega-3 oils.  CSIRO has been actively investigating these oils in areas including the recent focus on the development of new sources of LC omega-3 oils from terrestrial land plants and the characterization of novel marine oils for enhanced utilization of fisheries by-products. We provide an overview of chromatographic techniques and analytical methodologies developed, refined and employed by CSIRO in a range of omega-3 oil activities.  Techniques include: Thin-Layer Chromatography with flame ionization detection (TLC-FID) for lipid class characterisation and quantification, rapid GC methods for the analysis of ω3 LC-PUFA, argentation thin layer chromatography (Ag+-TLC) and solid phase extraction (Ag+-SPE) for separation of PUFA based on degree of unsaturation. Various HPLC techniques used include monolithic normal phase chromatography for rapid separation of lipid classes and preparative RP-HPLC for the purification of individual polyunsaturated fatty acids as fatty acid methyl esters (FAME). Several novel PUFA including very long-chain PUFA (28:7ω6 and 28:8ω3) in dinoflagellates and several odd-chain PUFA in marine thraustochytrids, including 21:5ω5 and 21:4ω7) were isolated and identified by GC-MS of their 4,4-dimethyloxazoline (DMOX) derivatives. LC-MS techniques were developed for the characterization and quantification of lipids including triacylglycerols (TAG), diacylglycerols (DAG), monoacylglycerols (MAG) and Acyl-CoA to assess PUFA and oil biosynthesis and to characterise GM land plant derived oils. Limitations in using LC-MS for quantification of lipids and differences in response are highlighted.