Igor Stimac's side were utterly dominant at the Gradski vrt stadium as goals from Mario Mandzukic and Eduardo moved Croatia onto 10 points from four matches, with Belgium and Scotland kicking off later.
Boasting an impressive record of just one home defeat in 46 competitive fixtures since claiming independence, the hosts immediately had Wales on the back foot.
Lewis Price, deputising once again for the injured Wayne Hennessey, was forced into a sprawling save on 10 minutes to deny veteran Darijo Srna, the start of what would prove to be a busy evening for the Crystal Palace goalkeeper.
Wales secured their first victory under Chris Coleman's reign against Scotland (2-1) on Friday but struggled to even escape their own half in the opening exchanges as Price was twice called upon to deny Ivan Rakitic.
The saturated surface in Osijek hindered both sides ability to knock the ball around freely, and indeed proved a factor in Croatia's opening goal.
A weak backpass from Ashley Williams forced Price into a rushed clearance that ricocheted off the charging Mandzukic, leaving the Bayern Munich striker with the simplest of finishes on 27 minutes.
Eduardo should have punished Wales further after some absent marking left him free in the box, but the Shakhtar Donetsk frontman failed to connect with Ivan Strinic's teasing cross from the right.
The former Arsenal man was then denied again twice in quick succession, a desperation block from Ben Davies then a reflex save from Price keeping him at bay.
With Gareth Bale the only player to have scored since Coleman took charge in January, it was all too evident how reliant Wales were on the Spurs star for any sort of attacking threat.
Aside from a speculative long-range effort and a couple of dangerous set-pieces, there was little for the Croatian backline to concern themselves with, and Mandzukic should have doubled the home side's lead 10 minutes into the second half when he was played clean through.
Price spread himself well to prevent Wales from falling further behind, but it was just a temporary stay of execution as Eduardo poked in a rebound following a corner a minute later.
The visitors had a chance to reduce the arrears but Andy King's powerful header was straight at Pletikosa.
Another brilliant instinctive save from Price saw him tip Eduardo's header onto the crossbar before smothering the ball after a chaotic goalmouth scramble.
A sensational solo run from Bale almost provided Wales with a consolation goal just before time, but Pletikosa got down well to push away his angled shot to keep the clean sheet intact and give Croatia their third win in four matches.