I don't know how to eloquently describe this (English is not my primary language) so I'll jump straight ahead with description/repro of my problem;
make sure Opus is in dual pane mode, open C:\Program Files\ in left pane and Desktop in right pane
set right pane as active
close Opus (regular X)
open some folder from Desktop or Start Menu (such as My Documents or similar)
observe that the tab in the left pane is "overwritten" with new location (instead of new tab being opened - option Open external folders in a new tab IS checked on my end) and as a bonus this happened in left pane even though right one is set as active
I would very much welcome solution for this situation(s) as it bit me in the ass numerous times
Right below the option you mention is another one: Preferences / Launching Opus / Explorer Replacement / Open all Default Lister tabs when opening a new window. I think you just need to turn that on.
If the whole page is missing then Opus doesn't think Explorer Replacement is turned on in USB mode.
That would happen if the ini file was no longer set to enable it. I think it may also happen if you run X:\DOPUS\dopus.exe instead of X:\DOPUS.EXE (where X:\ is the USB stick) as I think it's that stub exe which parses the ini file (although I may be mis-remembering from when I looked earlier today).
Are you running the USB Opus on a machine that already has Opus installed, with Explorer Replacement already turned on for the installed copy?
In that situation (although I didn't realise it until now, or I would've mentioned it earlier; apologies), the USB copy will not enable Explorer Replacement for itself as doing so could trash the registry settings of the main HDD install. (And it's probably not necessary anyway as the registry settings will run the main HDD install, which will realise the USB copy is already running and pass the request on to it. There might be problems if the two versions of Opus are really different and one tries to send parameters the other doesn't understand, but generally it should work. Of course, outside of testing, there's rarely a reason to run a USB copy of Opus on a machine with an HDD copy.)
So, if you launch the main HDD copy of Opus and turn off Explorer Replacement in it, then exit that copy and launch the USB copy, it will respect the flag in the DOPUS.INI file.
It's possible Opus thinks Explorer Replacement is enabled on the machine due to some registry values which didn't get cleaned up when the USB version exited (e.g. it could happen if power is cut to the machine when the USB version is running in Explorer Replacement mode).
If you look at HKEY_CLASSES_ROOT\Folder\shell using RegEdit, what's the b[/b] value set to, if anything? If it's "openindopus", exit Opus and wait a few seconds, then delete the value and it should make things work again.
(Alternatively, there's a .reg file in How to manually remove Explorer Replacement settings which will do the same and tidy up a couple of other things. Be sure to exit Opus and wait a few seconds before double-clicking it.)