Objective In this study, we designed and synthesized four novel Ga-68-radiolabeled compounds ([Ga-68]DN-3, [Ga-68]DN-4, [Ga-68]NN-3, and [Ga-68]NN-4) composed of a nitroimidazole and two types of bifunctional chelates (DOTA or NOTA) via several alkyl linkers of different length. Then, we evaluated their properties as hypoxia imaging probes for positron emission tomography (PET) compared with conventional compounds ([Ga-68]DN-2 and [Ga-68]NN-2).Methods The precursors of Ga-68-radiolabeled compounds were synthesized through a two-step reaction, and then reacted with (GaCl3)-Ga-68 to be Ga-68-radiolabeled compounds. FaDu cells were treated with Ga-68-radiolabeled compounds and then incubated under normoxic (21% O-2) or hypoxic (1% O-2) conditions. The radioactivity of these cells was measured 2 h after incubation. The biodistribution and PET/CT imaging of Ga-68-radiolabeled compounds in FaDu-bearing Balb/c nude mice were evaluated 2 h after intravenous injection.Results The Ga-68-radiolabeled compounds were synthesized with radiochemical purities over 95%. In the in vitro study, the levels of Ga-68-radiolabeled compounds were significantly higher in hypoxic cells than in normoxic cells. In hypoxic cells, the compounds we designed in this study demonstrated higher accumulation than the conventional compounds. In the in vivo biodistribution study, [Ga-68]DN-3 exhibited the highest accumulation in tumor. In the in vivo PET/CT imaging study, the tumor tissues of the FaDu-xenografted mice were visualized at 2 h after intravenous administration of Ga-68-radiolabeled compounds.Conclusions Our study suggested that the length of the linkers connecting nitroimidazole to a bifunctional chelate affect PET imaging of hypoxic tumors with Ga-68-radiolabeled compounds.