Low birth weight and preterm births contribute significantly to high neonatal mortality rates globally; resulting in 35% of the world’s 3.1 million annual deaths. Addressing challenges associated with neonatal deaths in Kenya has great potential through use of feasible, low-cost care practices leading to reduction of low birth weight and preterm birth fatalities. The objective of the study was to assess knowledge and practices recommended for care of preterm and low birth weight infants while in hospital and after discharge. A prospective cohort study was conducted in the New Born Unit of Kenyatta National Hospital. A structured questionnaire, key informant interviews and observations were used to collect data on socio-demographic characteristics, knowledge and practices related to care of the infants. In hospital 157 mother-infant dyads were enrolled using exhaustive sampling; during follow-up 124 (79%) of the sampled dyads participated. Knowledge on thermoregulation was insufficient with 52.9% of the mothers reporting that they had not been counselled on kangaroo mother care and 54.1% on ensuring their infant was in a warm room. Majority of the mothers (91.1%) lacked knowledge that pre-lacteal feeds are detrimental to the infant. A weak association existed between gestational age and maternal knowledge scores (r = 0.214).