Different genetic and environmental factors like altitude, temperature, light radiation, etc. affect the production of phytoconstituents like ascorbic acid, phenol, flavonoid, tannin, etc. So, it is necessary to evaluate phytoconstituents quantitatively and qualitatively because of their different medicinal values. This study is aimed at evaluating and comparing ascorbic acid concentration, total phenol, and flavonoid content in citrus species grown in different places in Western Nepal. For this, fruit collected from Lamjung and Nawalparasi was peeled, and collected juice was extracted with ethanol. Ascorbic acid was estimated using titration with dichlorophenolindophenol dye, and total phenol was estimated using Folin reagent; whereas, flavonoid content was determined by aluminum chloride colorimetric assay. The result showed the maximum ascorbic acid concentration in the fruit juice of Citrus maxima collected from Lamjung, i.e., 2.98 ± 0.161   mg / 100   ml , and the least concentration was recorded on extract of Citrus limon collected from Nawalparasi, i.e., 1.005 ± 0.205   mg / 100   ml . The total phenol content was comparatively higher in Citrus maxima and Citrus aurantifolia collected from Lamjung, i.e., 12.48 ± 0.40   mg GAE/gm, respectively. Flavonoid content was comparatively higher in Citrus maxima collected from Nawalparasi, i.e., 484.466 ± 3.055   mg QE/gm. This study showed a variation in the concentration of chemical constituents between different places in Western Nepal. This might be due to differences in climatic conditions, environmental factors, altitude, temperature, etc. This study helps to reflect the best-suited altitude for commercial cultivation of the citrus species as these phytochemicals have different medicinal values. Also, this study can help the food industry to use an alternative source of synthetic antioxidant such as ascorbic acid, phenol, and flavonoid. And local people will economically benefit by the commercial cultivation of citrus fruits.