Root canal filling aims at eliminating empty spaces into the root canal system using biologically compatible materials. Three-dimensional root canal obturation must prevent or minimize the reinfection caused by microorganisms' leakage. This study aimed at evaluating whether fluorophore (Rhodamine or Fluo-3) influences the CLSM images of intratubular penetration of four endodontic sealers. Eighty bovine teeth were prepared using K files up to a size #70 and irrigated with 2.5% sodium hypochlorite. All roots were divided into eight groups (n = 10) according to the sealer and fluorophore used: AH Plus/Rhodamine, AH Plus/Fluo-3, Sealer Plus/Rhodamine, Sealer Plus/Fluo-3, Sealer Plus BC/Rhodamine, Sealer Plus BC/Fluo-3, Endosequence/Rhodamine, and Endosequence/Fluo-3. All roots were filled using cold lateral compaction technique. After 7 days, the roots were transversely sectioned, and three slices, one of each canal third, were obtained. Intratubular penetration was evaluated using CLSM. Sealer Plus BC/Rhodamine and Endosequence BC/Rhodamine presented higher intratubular penetration than AH Plus/Fluo-3 and Sealer Plus/Fluo-3 (p ˂ .05). The intragroup analysis showed similar intratubular penetration, regardless of the root third, except for the apical third in AH Plus/Fluo-3 and Sealer Plus BC/Fluo-3 groups. The type of fluorophore influences the calcium silicate sealers' tubular penetration but not of epoxy resin-based ones using CLSM. Bioceramic sealers should not be used associated with Rhodamine for CLSM evaluation. RESEARCH HIGHLIGHTS: The type of fluorophore influences the calcium silicate sealers' tubular penetration but not of epoxy resin-based ones when CLSM is used for assessment. Bioceramic sealers should not be used associated with Rhodamine.