Persistent atrial fibrillation (AF) is not effectively treated due to a lack of adequate tools for identifying patient-specific AF substrates. Recent studies revealed that in 30–50% of patients, persistent AF is maintained by localized drivers not only in the left atrium (LA) but also in the right atrium (RA). The chamber-specific atrial wall thickness (AWT) features underlying AF remain elusive, though the important role of AWT in AF is widely acknowledged. We aimed to provide direct evidence of the existence of distinguished RA and LA AWT features underlying AF drivers by analysing functionally and structurally mapped human hearts
ex vivo
. Coronary-perfused intact human atria (
n
= 7, 47 ± 14 y.o.; two female) were mapped using panoramic near-infrared optical mapping during pacing-induced AF. Then the hearts were imaged at approximately 170 µm
3
resolution by 9.4 T gadolinium-enhanced MRI. The heart was segmented, and 3D AWT throughout atrial chambers was estimated and analysed. Optical mapping identified six localized RA re-entrant drivers in four hearts and four LA drivers in three hearts. All RA AF drivers were anchored to the pectinate muscle junctions with the crista terminalis or atrial walls. The four LA AF drivers were in the posterior LA. RA (
n
= 4) with AF drivers were thicker with greater AWT variation than RA (
n
= 3) without drivers (5.4 ± 2.6 mm versus 5.0 ± 2.4 mm,
T
-test
p
< 0.05;
F
-test
p
< 0.05). Furthermore, AWT in RA driver regions was thicker and varied more than in RA non-driver regions (5.1 ± 2.5 mm versus 4.4 ± 2.2 mm,
T
-test
p
< 0.05;
F
-test
p
< 0.05). On the other hand, LA (
n
= 3) with drivers was thinner than the LA (
n
= 4) without drivers. In particular, LA driver regions were thinner than the rest of LA regions (3.4 ± 1.0 mm versus 4.2 ± 1.0 mm,
T
-test
p
< 0.05). This study demonstrates chamber-specific AWT features of AF drivers. In RA, driver regions are thicker and have more variable AWT than non-driver regions. By contrast, LA drivers are thinner than non-drivers. Robust evaluation of patient-specific AWT features should be considered for chamber-specific targeted ablation.