Abstract: Numerous fluorescent marker lines are currently available to visualize microtubule (MT) architecture and dynamics in living plant cells, such as markers expressing p35S::GFP-MBD or p35S::GFP-TUB6. However, these MT marker lines display obvious defects that affect plant growth or produce unstable fluorescent signals. Here, a series of new marker lines were developed, including the pTUB6::VisGreen-TUB6-expressing line in which TUB6 is under the control of its endogenous regulatory elements and eGFP is replaced with VisGreen, a brighter fluorescent protein. Moreover, two different markers were combined into one expression vector and developed two dual-marker lines. These marker lines produce bright, stable fluorescent signals in various tissues, and greatly shorten the screening process for generating dual-marker lines. These new marker lines provide a novel resource for MT research.