Glucagon-like peptide-1 receptor (GLP-1R) targeting using radiolabeled exendin is a promising approach to noninvasively visualize and determine beta cell mass (BCM), which could help to unravel the pathophysiology of diabetes. However, saturation of the GLP-1R on beta cells occurs at low peptide doses, since the number of receptors expressed under physiological conditions is low. Therefore, tracers with high specific activities are required to sensitively image small variations in BCM. Here, we describe a novel exendin-3-based radiotracer with multiple chelators and determine its potential for in vivo beta cell imaging. Exendin-3 was modified by adding six lysine residues C-terminally conjugated with one, two, or six DTPA moieties. All compounds were labeled with In and their GLP-1R affinity was determined in vitro using GLP-1R expressing cells. The in vivo behavior of the In-labeled tracers was examined in BALB/c nude mice with a subcutaneous GLP-1R expressing tumor (INS-1). Brown Norway rats were used for SPECT visualization of the pancreatic BCM. Addition of six lysine and six DTPA residues (hexendin(40-45)) resulted in a 7-fold increase in specific activity (from 0.73 GBq/nmol to 5.54 GBq/nmol). IC values varied between 5.2 and 69.5 nM. All compounds with two or six lysine and DTPA residues had a significantly lower receptor affinity than [Lys (DTPA)]exendin-3 (4.4 nM, p < 0.05). The biodistribution in mice revealed no significant decrease in pancreatic uptake after addition of six lysine and DTPA molecules. Hexendin(40-45) showed a 6-fold increase in absolute In uptake in the pancreas of Brown Norway rats compared to [Lys (DTPA)]exendin-3 (182.7 ± 42.3 kBq vs 28.8 ± 6.0 kBq, p < 0.001). Visualization of the pancreas on SPECT was improved using hexendin(40-45), due to the higher count rate, achieved at the same peptide dose. In conclusion, hexendin(40-45) showed an improved visualization of the pancreas with SPECT. This tracer holds promise to sensitively and specifically detect small variations in BCM.