Can I set a different markup for my sub agents than the one I use for my walk in customers?

Yes, most travel agency software lets you keep more than one markup rule and applies the right one on its own. The system looks at who is logged in when the search runs, so a sub agent signing in with their own account sees the fare with the sub agent markup on it, while a booking you make yourself at the counter picks up your retail markup instead. You set the rules once in the back office and they apply to every search from then on, without anyone having to remember to add anything by hand.

How finely you can slice those rules depends on your plan and on how your account has been set up. In most cases you can go past a single flat rule and set the markup by product, so flights carry one rate and hotels another, or set it as a percentage rather than a fixed amount, or give one large sub agent their own rate that is separate from the rest. Some suppliers also allow their own negotiated rates to be handled differently, so it is worth asking before you assume a rule will reach everything.

One thing to keep in mind is that a markup sits on top of whatever fare the supplier sends back, so it moves up and down with that fare. Any commission the supplier pays you is a separate matter and does not show inside the markup, which means your real earning on a booking can be more than the markup alone suggests.

Before you go live, run the same search twice, once from your own login and once from a test sub agent login, and compare the two prices to be sure each rule is landing where you expect.