Abscisic acid (ABA) is a plant hormone regulating the essential physiological processes of plants and plant responses to various environmental stresses. Recent studies revealed that the key enzymes involved in ABA biosynthesis were zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenases (NCED), short-chain dehydrogenase/reductase (SDR), and ABA-aldehyde oxidase (AAO). In this study, we cloned 12 unique genes potentially involved in ABA synthesis, including 5 of BpNCEDs, 1 of BpAAO, and 6 of BpZEPs from birch leaves. We analyzed the time-course expression of these 12 genes during the phase from May to August by real time reverse transcriptase-polymerase chain reaction (RT-PCR). Further, we determined birch endogenous ABA content during this period by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results indicated that ABA level significantly (P < 0.05) increased during the early stages of development and ABA was present in birch leaves throughout the studied period, suggesting that endogenous ABA is necessary for the normal growth and development of plants. The correlation between the expression of above-mentioned genes and ABA levels was analyzed by Pearson correlation analysis. The results revealed that the expression of three genes, namely, BpNCED4, BpNCED5 and BpZE6 significantly (P < 0.05) correlated with the ABA level. Therefore, these three genes may play important roles in ABA biosynthesis.