Jason Puncheon drilled the ball across Adam Federici in the 61st minute for the only goal of the game, having seen a first-half header harshly chalked off for a foul on the Reading keeper.
Brian McDermott's side posed little threat, with their best chance coming in first-half stoppage time when Hal Robson-Kanu headed against the post.
Southampton made two changes to the side that started at Anfield, Kelvin Davis replacing the injured Paulo Gazzaniga and Jay Rodriguez in for Gaston Ramirez, who had spent much of the week in Uruguay following a family bereavement. Reading were unchanged form the side that started the defeat to Manchester United last week.
The home side looked the more likely to open the scoring early on, but they failed to really hurt Reading despite their vastly superior possession.
Puncheon proved to be a nuisance for Southampton, putting in several decent crosses in the opening stages with Morgan Schneiderlin having the best chance when he was just unable to get on top the ball at the near post.
Adam Lallana, skippering the side despite the recall of club captain Davis, showed why he is said to be attracting a lot of interest from other clubs, driving a half-volley chance straight at Federici.
Jay Tabb was Reading's unlikely goal threat, making a couple of breaks from midfield and shooting straight at a largely underemployed Davis.
But the home side looked to have broken the deadlock after 33 minutes when Puncheon rose at the far post to nod a corner home. Referee Jon Moss immediately blew for an infringement, penalising Maya Yoshida for a push on Federici, but the decision looked a harsh one.
However the visitors nearly made amends for a toothless first half in stoppage time when Robson-Kanu's header struck the left-hand upright. Davis was rooted to the spot, but a goal for Reading at this stage would have been hard on Southampton.
Lallana's impressive first half display ended early when he limped off with a knee injury, but Southampton shrugged off the loss of their talismanic midfielder and continued to look much the better side after the break.
Puncheon had already forced Federici into a good save when he finally gave his side the lead they had deserved. Rickie Lambert fed Nathaniel Clyne, and the right back in turn passed to Puncheon, who broke in from the right to shoot low across Federici into the bottom corner of the net.
Jobi McAnuff curled a shot wide of Davis' left-hand post in response, and Robson-Kanu put a weak effort on the turn straight at the Southampton keeper, but it was the hosts who continued to look likelier to score.
Puncheon drilled a low shot from long range wide before substitute Guly Do Prado found the roof of the net from distance. The Brazilian had another chance in the closing stages, but was denied by a fine save from Federici down to his left.
Saints continued to dominate and were good value for their win, whereas Reading ended the game looking at a long, hard winter with a crucial trip to fellow strugglers Sunderland looming on Tuesday.
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