•Photobioreactor, open pond and flask cultivation of Chlorella sp. was compared.•Photobioreactor showed higher biomass yield, productivity and chlorophyll content.•All cultivation systems showed higher lipid & neutral lipids from stationary phase culture.•Similar fatty acids pattern from cultures grown in all cultivation systems was seen.•γ-Linolenic acid (14–15%) and palmitic acid (11–13%) are prevalent fatty acids. Microalgae are the promising feedstock for biodiesel production than other first and second-generation feedstocks. Generating huge biomass sustainably per unit volume per day is vital to the continuous utilization of algal feedstock for sustainable biodiesel production. But, cultivation and harvesting of microalgae are the significant cost drivers and the commonly used cultivation systems for microalgae are outdoor pond and photobioreactor. In this study, small scale photobioreactor, outdoor open pond and Erlenmeyer flask based cultivation of Chlorella sp. and harvesting at log phase and stationary phase for biomass, lipids towards FAME (fatty acid methyl ester) production was investigated. Photobioreactor grown Chlorella sp. showed higher growth in terms of biomass yield, biomass productivity and chlorophyll content. In the case of lipid content, Chlorella sp. unveiled a similar percentage of lipid about 20% (log phase) and 24% (stationary phase) irrespective of the cultivation systems tested. Non-polar lipid percentage 46% in log phase, while in stationary phase, Chlorella sp. showed higher levels of non-polar lipids at about 66% in photobioreactor and open pond systems. A similar pattern of fatty acids was seen from cultures grown in all three cultivation systems and among identified fatty acids, γ-linolenic acid is prevalent in the range between 14 and 15% followed by palmitic acid in the range between 11 and 13%.