Valencia's off-the-field issues have overshadowed their on-field exploits since Marcelino's sacking last week, but Albert Celades' side bounced back from their 5-2 defeat to Barcelona with a resilient performance at Stamford Bridge.
With Jasper Cillessen in fine form and Mason Mount's injury having compounded Chelsea's frustrations, the Blues' frailties were exposed once more when Rodrigo Moreno prodded in Dani Parejo's free-kick with16 minutes remaining.
VAR came to Chelsea's rescue when the referee decided to award the hosts a spot-kick for a Daniel Wass handball, but substitute Barkley - who overruled Willian to take the penalty - struck the bar.
Barkley steps up to take it but it skims the bar and heads over.— Chelsea FC (@ChelseaFC) September 17, 2019
0-1 [87'] #CHEVCF
Fresh from his hat-trick against Wolves, Tammy Abraham should have done better in the sixth minute when he latched onto Cesar Azpilicueta's cross - Cillessen making the save.
Chelsea were dealt a blow soon after, Mount's Champions League debut cut short as he failed to overcome an ankle injury inflicted by Francis Coquelin's robust challenge.
Willian's volley drew a brilliant stop out of Cillessen on the stroke of half-time and Valencia's goalkeeper had more work to do when he kept out Marcos Alonso's free-kick.
But just after Lampard had gambled in throwing on Olivier Giroud for Kurt Zouma, Valencia struck.
Having stole a march on his markers, Rodrigo just managed to make enough contact on Parejo's superb pass.
Chelsea's fortunes looked to have turned with four minutes remaining when referee Cuneyt Cakir handed them a lifeline having looked at the touch-line screen, but Barkley failed to keep his composure.
What does it mean? Injuries Mount up for Lampard
To add injury to insult, Lampard - who was already without Antonio Rudiger, Emerson and N'Golo Kante heading into Tuesday's match - could now be missing influential midfielder Mount for Liverpool's trip to Stamford Bridge on Sunday.
Rodrigo comes alive when it matters
Valencia's front two had hardly tested Kepa Arrizabalaga prior to Rodrigo's chance, but the Spain international made no mistake with a clinical finish.
Barkley's confidence misplaced
Barkley had only been on the pitch seven minutes when he stepped up to take the spot-kick, even though Willian seemed to want it, and there is sure to be an inquest in the Chelsea dressing room as to why the substitute pulled rank after a dismal attempt.
Key Opta Facts
- This was only Chelsea's second defeat in their last 42 Champions League group stage games at Stamford Bridge (W30 D10 L2), with the other against Basel in 2013.
- Frank Lampard became the first Chelsea manager to lose his first Champions League match in charge – 10 of the previous 11 had won, while Gianluca Vialli drew with Milan in 1999.
- Valencia registered only their second away Champions League win in England (W2 D7 L3), with their first since winning 1-0 at Liverpool in October 2002.
- Rodrigo netted only his third Champions League goal in his 25th appearance in the competition and his first in 10 appearances for Valencia.
- Barkley was the first Englishman to take a penalty for Chelsea since Frank Lampard in April 2014 against Stoke City – both players missed their penalties.
- Chelsea became the first team since Liverpool in December 2008 against PSV (Darby, Kelly, Spearing) to hand three Champions League debuts to Englishmen aged 21 or younger in the same game (Abraham, Mount, Tomori).
The big games come thick and fast for Chelsea, with Premier League leaders Liverpool coming up, while Valencia face Leganes in LaLiga.