The poor quality of available feed resources is the major limiting factor for livestock production in Ethiopia. The aim of this study was to determine and compare the chemical composition of major feed resources in three agro-ecological zones (AEZs) of the Gera District, southwest Ethiopia. Three representative samples of natural pasture (Cynodon dactylon, Pavonia schimperiana Hochst and Rhynchosia ferruginea), three indigenous fodder trees and shrubs (IFTSs) (Erythrina abyssinica, Vernonia amygdalina and Maytenus undat), two cultivated forages (Pennisetum purpureum and Pennisetum pedicellatum) and five crop residues (Hordeum vulgare (barley), Zea mays (maize), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Eragrostis tef (Teff)) were randomly and separately collected from the Highland (HL), midland (ML) and lowland (LL) AEZs. The samples were analyzed for ash, dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), ether extract (EE), and acid detergent lignin (ADL) content using standard analytical methods. The results showed that AEZ significant (P < 0.05) affected the chemical composition parameters of most of the feed samples. Regardless of the feed sample and AEZs, the mean DM, CP, ash, EE, NDF, ADF, ADL and CF content varied from 89.17% in Pennisetum purpureum to 92.22 ± 0.51 in Erythrina abyssinica, 2.90 ± 0.22% in Triticum aestivum to 10.70 ± 0.52 in Vernonia amygdalina, 7.24 ± 0.19% in Sorghum bicolor to 13.25 ± 0.51% in Pavonia Schimperiana Hochst, 1.33 ± 0.04% in Triticum aestivum to 2.39 ± 0.15% in Maytenus undata, 57.65 ± 1.19% in Erythrina Abyssinica to 79.16 ± 1.04% in Triticum aestivum, 34.57% in Pennisetum purpureum to 59.41 ± 0.98% in Sorghum bicolor, 8.15 ± 0.62% in Rhynchosia ferruginea to 12.65 ± 0.57% in Triticum aestivum and 37.51% in Pennisetum pedicelatum to 69.93 ± 0.65% DM in Triticum aestivum, respectively. It was observed that IFTSs had the highest CP and the lowest NDF, implying their potential for protein supplement, whereas crop residues had the lowest CP and the highest NDF, indicating the need for their treatment using urea or supplementing animals with protein source feeds, especially during the dry season. In conclusion, AEZ has an influence on the chemical composition traits of feed resources attributed to its effects on the microclimate conditions that affect plant growth and maturation in the study sites.