Rootstocks have a significant influence on adaptation to biotic and abiotic stress conditions and quality of fruit. So, choice of rootstock for a specific growing region is an important decision for a grower to make when establishing a commercial citrus orchard. This study was undertaken to assess effects of rootstock and location on quality of orange fruit. The study was conducted over two consecutive years (2017/18 and 2018/19). Scions from the Gunda Gundo orange grove were grafted on rough lemon and sour orange rootstocks and planted in Tigray and Afar, Ethiopia at four sites (Abala, Agulae, Adiha and Rama). The experiment was laid out in Randomized Complete Block Design (RCBD) with four replications. Data on different physico-chemical and bioactive compounds were collected and analyzed using R software (version 4). Compared to sour orange, rough lemon rootstock revealed higher fruit mass (151.96 g, 195.38 g), fruit length (62.27 mm, 68.88 mm) and fruit diameter (65.58 mm, 71.3 mm), in 2017/18, 2018/19, respectively. Among the four locations (Abala, Agulae, Adiha and Rama), higher juice percentage was found in Adiha (43.47％in 2017/18) and Abala (49.87％, in 2018/19). In 2017/18 higher total soluble solid (TSS, 13.87％), TSS/acid ratio (19.18) and fruit rag (32.77％), were obtained at the Rama site. On the one hand, higher vitamin C (826.6 mg·kg? 1) and total phenolic content (208 mg·kg? 1 GAE) were recorded at Agulae than Abala, Adiha and Rama sites from sour orange rootstock. High TSS, TSS/acid ratio and antioxidant were recorded from orange fruits at Rama site. This was expected to have resulted from hot climate conditions at the site. Besides, at a relatively cooler condition of the Agulae site, high phenolic, reduced ferric power and high vitamin C were recorded. Rough lemon rootstock improved fruit size, while rootstock did not affect the contents of TSS, TSS/acid ratio across the sites in both harvesting years.