Substrate functionalization is of great importance in successfully manipulating flows and liquid interfaces in microdevices. Herein, we propose an alternative approach for spatial patterning of wettability in a microcapillary tube. The method combines a photolithography process with self-assembled monolayer formation. The modified microcapillaries show very sharp boundaries between the alternating hydrophilic/hydrophobic segments with an achieved smallest domain dimension down to 60 μm inside a 580 μm inner diameter capillary. Our two-step method allows us to pattern multiple types of functional groups in an enclosed channel. Such structures are promising regarding the manipulation of segmented flows inside capillaries.