Gareth Anscombe and Tom Curry suffered injuries just six weeks before the Rugby World Cup starts as an experimental England side prevented Wales from going top of the rankings with a 33-19 win at Twickenham.
Australia's Bledisloe Cup victory on Saturday opened the door for the Six Nations champions to replace New Zealand as the top-ranked side in the world for the first time, but they were denied by a fired-up England.
Wales also have a big concern over the fitness of Anscombe, the fly-half limping off in the first half after damaging his left knee in Alun Wyn Jones' Welsh record 135th Test.
England lost Curry to what appeared to be an arm injury in the first half on the eve of Eddie Jones' squad announcement for the tournament in Japan.
Tries from man of the match Billy Vunipola, Joe Cokanasiga and Luke Cowan-Dickie, 15 points from the boot of captain George Ford and an Elliot Daly drop goal gave England a morale-boosting victory, with debutants Willi Heinz and Lewis Ludlam among those to impress along with the fit-again Anthony Watson.
Gareth Davies scored a superb solo try, while George North and Jones also crossed, but a strong Wales side - who will get the chance to gain revenge in Cardiff next weekend - looked rusty and could not match England's intensity.
Wales did not know what had hit them as England made a blistering start, the brilliant Vunipola crashing over four minutes in with Heinz dictating and the forwards overpowering the Wales pack.
Cokanasiga bulldozed his way over from short range before Davies produced a moment of magic to get a flat Wales side on the board, bursting his way through from the back of a scrum at great speed and capitalising on slack defending to go under the posts.
England lost Curry before Anscombe was helped off in pain and there was more woe for Wales when Ken Owens' poor throw at a lineout sailed over captain Jones, gifting Cowan-Dickie a first Test try.
Ford converted that score just before the break and made it four out of four from the tee with a penalty early in the second half to put England 17 points to the good.
Wales finally found another gear and were rewarded with quickfire scores from North and Jones, but two more Ford penalties and a strike from Daly took the world number one status beyond Wales.