Biodiesel from jatropha, neem and cottonseed were produced and their compositions were analyzed using gas chromatography–mass spectroscopy. The results revealed that the jatropha, neem and cottonseed oils are reliable sources of biodiesel and are renewable, and hence have a potential to partially meet energy demands in an eco-friendly manner. The biodiesels produced from jatropha contained 91.97% methyl ester, and that produced from neem contained 70.2% methyl ester, whereas that produced from cotton contained 89.75% methyl ester.The greater proportion of prolific ester in the jatropha biodiesel is mono-saturated methyl octa-decanoate which contributes 53.44% by volume. This ester has the tendency of assigning stability to biodiesel. For neem-based biodiesel, the profile evaluation revealed that octadecanoic acid methyl ester is the dominant compound in the mixture with 54.69% by volume. For cotton, the profile revealed that the methyl octadecenoate (C₁₉H₃₆O₂) is the predominant compound in the mixture, having the highest percentage of 17.72%. The physico-chemical analyses of the three biodiesels has indicated that the three conform with the American Society for Testing and Materials (ASTM) standards for specific gravity, viscosity, acid value, cold flow properties and sulfur content, and hence are suitable for use in compression ignition engines. Statistical analyses confirmed that there was no significant difference between the experimental values of all the physico-chemical properties investigated; therefore, the experimental data is validated.