This dataset contains morphometric and isotopic data for American alligators (Alligator mississippiensis) that were sampled from within four freshwater (FW) wetlands and one estuarine (EST) wetland within the Greater Everglades ecosystem. Due to their compartmentalization and varied habitat and water regimes, there were multiple sampling sites within two of the FW wetlands. The five wetlands were the Arthur R. Marshall Loxahatchee National Wildlife Refuge (LOX), Big Cypress National Preserve (BICY), Water Conservation Area 3 (WCA3; includes WCA3A-Tower (TW), WCA3A-Holiday Park (HD), WCA3A-North HWY 41 (N41) and WCA3B-3B sites), Everglades National Park (ENP-FW; includes ENP-FC, ENP-SS, and ENP-NESSE sites), and an estuary system within ENP (ENP-EST). The subtropical climate of south Florida has hot humid summers, mild winters, and surface hydrology that is managed for flood protection, water supply, and ecological needs. Alligators were sampled between South Florida Water Management District Water Years (WY; May 1 of the previous year to April 30 of the reporting year) 2013-2020 during the spring dry seasons (February-May) and fall wet seasons (September–November) within the five wetlands. Immediately following capture, we collected ~1.5-2.0 mL of whole blood via the post-occipital venous sinus or the ventral coccygeal vein with a sterile, 20-gauge 5.0 mL capacity syringe. During WY 2013-2016 we also collected plasma fractions by immediately centrifuging blood in the field using a portable centrifuge. In spring of WY 2016 both plasma and whole blood were collected. From fall of WY 2016 through spring of WY 2020 logistical constraints prevented us from collecting plasma fractions, thus we were only able to collect whole blood samples. After collection, we placed both plasma fractions and whole blood samples into individually labeled, sterile Corning Cryovials. We stored the vials on ice in the field until they were transferred into a -20 °C freezer in the laboratory later that same night.